Differentiating induced pluripotent stem cells into glucose-responsive, insulin-secreting progeny

ABSTRACT

This document provides methods and materials related to differentiating iPS cells into glucose-responsive, insulin-secreting progeny. For example, methods and material for using indolactam V (ILV) and glucagon like peptide-1 (GLP-1) to produce glucose-responsive, insulin-secreting progeny from iPS cells are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/340,161, filed Jul. 24, 2014, which is a continuation of U.S.application Ser. No. 13/553,064, filed Jul. 19, 2012 (Abandoned), whichclaims the benefit of U.S. Provisional Application Ser. No. 61/510,818,filed Jul. 22, 2011. The disclosures of the prior applications areconsidered part of (and are incorporated by reference in) the disclosureof this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved indifferentiating induced pluripotent stem (iPS) cells intoglucose-responsive, insulin-secreting progeny. For example, thisdocument relates to the use of indolactam V (ILV) and glucagon likepeptide-1 (GLP-1) to produce glucose-responsive, insulin-secretingprogeny from iPS cells.

2. Background Information

Stem cells are characterized by the ability of self-renewal anddifferentiation into a diverse range of cell types. The two broad typesof mammalian stem cells are embryonic stem

(ES) cells and adult stem cells. Adult stem cells or progenitor cellsreplenish specialized cells to repair or maintain regenerative organs.Most adult stem cells are lineage-restricted and generally referred toby their tissue origin, such as adipose-derived stem cells. ES celllines are derived from the epiblast tissue of the inner cell mass of ablastocyst or early morula stage embryos. ES cells are pluripotent andgive rise to derivatives of the three germinal layers, i.e., theectoderm, endoderm, and mesoderm.

SUMMARY

This document provides methods and materials related to differentiatingiPS cells into glucose-responsive, insulin-secreting progeny. Forexample, this document provides methods and material for using ILV andGLP-1 to produce glucose-responsive, insulin-secreting progeny from iPScells. As described herein, culturing iPS cells in the presence of acollection of agents that include ILV and GLP-1 can result in theproduction of glucose-responsive, insulin-producing cells. For example,an ILV and GLP-1-enriched pancreatogenic cocktail can be used underfeeder cell-free conditions to produce glucose-responsive,insulin-producing cells from human iPS cells. Autologous iPS cellderivation and iPS cell differentiation into insulin-producing cells canallow modeling of patient-specific disease pathogenesis and can lead topersonalized approaches for type 1 diabetes cell therapy withiPS-derived islet-like cells.

In general, one aspect of this document features a method for obtaininga population of glucose-responsive, insulin-secreting cells from apopulation of induced pluripotent stem cells.

The method comprises, or consists essentially of, culturing the inducedpluripotent stem cells with medium comprising indolactam V and glucagonlike peptide-1 under conditions to obtain the population ofglucose-responsive, insulin-secreting cells. The medium can lack serum.The medium can lack feeder cells. The medium can lack non-human feedercells. The induced pluripotent stem cells can be induced pluripotentstem cells that were obtained using one or more polypeptides or nucleicacid encoding the one or more polypeptides selected from the groupconsisting of a Oct3/4 polypeptide, a Sox family polypeptide, a Klffamily polypeptide, a Myc family polypeptide, a Nanog polypeptide, and aLin28 polypeptide. The induced pluripotent stem cells can be inducedpluripotent stem cells that were induced from somatic cells. The somaticcells can be selected from the group consisting of skin, lung, heart,stomach, brain, liver, blood, kidney, and muscle cells. The inducedpluripotent stem cells can comprise exogenous nucleic acid encoding ahuman Oct4 polypeptide, a human Sox2 polypeptide, a human Klf4polypeptide, and a human c-Myc polypeptide. The glucose-responsive,insulin-secreting cells can secrete greater than 50 pM of C peptide perhour when in culture in the presence of about 10 mM of glucose. Theglucose-responsive, insulin-secreting cells can secrete greater than 200pM of C peptide per hour when in culture in the presence of about 10 mMof glucose. The glucose-responsive, insulin-secreting cells can secretebetween about 50 and 250 pM of C peptide per hour when in culture in thepresence of about 10 mM of glucose. The glucose-responsive,insulin-secreting cells can be human cells. The medium can comprisegreater than 300 nM of indolactam V. The medium can comprise greaterthan 55 nM of glucagon like peptide-1. The culturing can be performedfor more than 25 days.

In another aspect, this document features a population ofglucose-responsive, insulin-secreting cells derived from inducedpluripotent stem cells, wherein the glucose-responsive,insulin-secreting cells are produced by culturing the inducedpluripotent stem cells with medium comprising indolactam V and glucagonlike peptide-1 under conditions that result in the formation of thepopulation of glucose-responsive, insulin-secreting cells. The mediumcan comprise greater than 300 nM of indolactam V. The medium cancomprise greater than 55 nM of glucagon like peptide-1. The culturingcan be performed for more than 25 days. The population ofglucose-responsive, insulin-secreting cells can secrete greater than 50pM of C peptide per hour when in culture in the presence of about 10 mMof glucose. The population of glucose-responsive, insulin-secretingcells can secrete greater than 200 pM of C peptide per hour when inculture in the presence of about 10 mM of glucose. The population ofglucose-responsive, insulin-secreting cells can secrete between about 50and 250 pM of C peptide per hour when in culture in the presence ofabout 10 mM of glucose. The glucose-responsive, insulin-secreting cellscan be human cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of Human iPS Clones from BJ and HCF Fibroblasts. (A)Lentiviral vector-mediated delivery of OCT3/4, SOX2, KLF4, and c-MYCresulted in iPS-like colony formation. (i) SNL feeder cells, (ii)uninfected HCF fibroblasts, (iii) HCF-derived iPS-like colony at twoweeks post-infection, (iv) iPS-like cells with high magnification. iPScells exhibited morphology similar to human ES cells, characterized bylarge nuclei and scant cytoplasm, (v) uninfected BJ fibroblasts, (vi) BJfibroblasts-derived iPS-like colony at two weeks after infection, (vii)image of a BJ-derived clone expanded on feeder cells, (viii) highmagnification image of BJ-derived clone. (B) Feeder-free generation ofhuman iPS cells allowed visualization of the early reprogramming events.(i) Uninfected BJ fibroblasts, (ii) an early stage iPS-like colony invector-transduced BJ cells one week after infection, (iii) highmagnification image of BJ fibroblast-derived iPS-like colony. (C)Morphology of iPS clones cultured under feeder-free conditions. BJ#SAwas established on SNL feeder cells, while HCF#1 and BJ#1 were derivedfeeder-free. (D) HCF#1, BJ#SA and BJ#1 cultured under feeder-freeconditions expressed high levels of alkaline phosphatase (AP).

FIG. 2. Feeder-free generation of human iPS cells allowed visualizationof the early reprogramming events. For feeder-free iPS generation, BJand MRCS fibroblasts were infected with lentiviral vectors expressingOCT4, SOX2, KLF4 and c-MYC (4 factor). After four days of infection,cells were replated on Matrigel coated plates. i. Uninfected BJfibroblasts, ii. BJ fibroblast-derived iPS-like colony at 7 days afterinfection, iii. BJ fibroblast-derived iPS-like colony at 8 days afterinfection, iv. The colony shown in (iii) was positive for alkalinephosphatase, v. MRC5-derived iPS-like colonies at 12 days after vectortransduction, vi. The same colonies shown in (v) were positive foralkaline phosphatase at 15 days after infection.

FIG. 3. Expression of Pluripotency-Associated Genes in Putative iPSClones. (A) and (B) HCF- and BJ-derived iPS clones were analyzed forexpression of pluripotency markers by immunostaining. HCF#1 and BJ#SAcells were positive for pluripotency markers SSEA4, TRA-1-60, TRA-1-81,OCT4, SOX2, KLF4, and NANOG, while no notable staining was observed forSSEA1. Cells were counterstained with 4′,6-diamidino-2-phenylindole(DAPI). Control (m) and Control (r); control cells treated withFITC-conjugated secondary antibodies against mouse IgG and rabbit IgG.Scale bars indicate 20 μm. (C) HCF- and BJ-derived iPS-like clones wereanalyzed for pluripotency-associated gene expression by RT-PCR. Totalcellular RNA from parental BJ and HCF fibroblasts and no template(water) samples were included as controls. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene transcript was amplified as an internal RNAcontrol.

FIG. 4. Spontaneous Differentiation of HCF- and BJ Fibroblast-derivediPS Cells into Cells of Three Embryonic Germ Layers. (A) In vitrodifferentiation of HCF#1, BJ#1, and BJ#SA clones in suspension cultureas embryoid bodies (EB) was followed by monolayer culture forspontaneous differentiation. HCF#1, BJ#1, and BJ#SA clones generated EBswith varying sizes. Cells of ectoderm, endoderm, and mesoderm lineageswere confirmed by beta III tubulin (green stain was used), FOXA2 (redstain was used), and CD31 (PECAM-1) (green stain was used),respectively. Cells were counterstained with DAPI. Scale bars on right50 um and left 20 μm. (B) Teratoma formation. iPS cells 500,000 wereinjected subcutaneously into athymic nude mice. Tumor growth wasdetected only from sites injected with iPS cells. After 3 months tumorswere harvested. Scale bar indicates 2 mm. (C) H&E staining of teratomasections demonstrated multiple lineages within the complex architectureof the tumor, including ectoderm (glandular tissue), endoderm (adiposetissue), and mesoderm (muscular tissue) tissues.

FIG. 5. Differentiation of Human iPS Cells into Pancreatic EndodermCells. (A) Schematic representation of the stepwise differentiationprotocol for generation of islet-like clusters from human iPS cells. DE,definitive endoderm; GTE, gut tube endoderm; PP, pancreatic progenitor;EN, endocrine hormone expressing cells; CYC, KAAD-cyclopamine; RA,all-trans retinoic acid; ILV, indolactam V; HGF, hepatocyte growthfactor; IGF, insulin like growth factor; and GLP-1, glucagon-likepeptide-1. (B) Induction of definitive endoderm cells. iPS cells weretreated with activin A and Wnt3a for one day, followed by activin A with2% FBS for two days. iPS-derived cells were immunostained withantibodies against SOX17 (green stain was used) and FOXA2 (red stain wasused). Cells were counterstained by DAPI. Bars indicate 20 μm. (C) Flowcytometric analyses of iPS-derived definitive endoderm cells.iPS-derived definitive endoderm cells were dissociated and stained withanti-SOX17 antibody. Staining with the secondary antibody alone was usedas a control. (D) iPS-derived definitive endoderm cells were treatedwith FGF10, CYC, RA, and ILV for induction of pancreatic endoderm andimmunofluorescence analysis was performed to detect, PDX1 (red stain wasused), NEUROD1 (red stain was used), and NGN3 (red stain was used).

FIG. 6. Differentiation of BJ#1 and BJ#SA Clones into DefinitiveEndoderm Cells. BJ#1 and BJ#SA cells were treated with activin A andWnt3a for one day, followed by activin A stimulation in the presence of2% FBS for two days for generation of definitive endoderm cells. (A)BJ#SA derived definitive endoderm cells were immunostained withantibodies against SOX17 (a green stain was used) and FOXA2 (a red stainwas used). (B) BJ#1 derived definitive endoderm cells were stained withantibody against SOX17. Cells were counterstained by DAPI. Bars indicate20 mm. (C) Flow cytometric analyses of iPS-derived definitive endodermcells. iPS-derived definitive endoderm cells were dissociated andstained with anti-SOX17 antibody. Staining with the secondary antibodyalone was used as a control.

FIG. 7. Efficient Differentiation of iPS Cells into Pancreatic EndodermCells. iPS-derived definitive endoderm cells were treated with FGF10,CYC, RA, and ILV for induction of pancreatic endoderm. On day 17 ofdifferentiation, immunofluorescence analysis was performed to detectpancreatic endoderm markers, PDX1 (a red stain was used) and NEUROD1 (ared stain was used) from BJ#SA-derived cells.

FIG. 8. Successful Differentiation of Human iPS Cells into PancreaticHormone-expressing Cells. (A) Induction of stage-specific pancreaticgenes through guided differentiation. RT-PCR analysis was performed todetermine the expression of key pancreatic genes at different stages ofdifferentiation. Undifferentiated human iPS cells (d0), definitiveendoderm cells after treatment with activin A and Wnt3a (d3), foregutendoderm cells induced with FGF10 and CYC (d9), pancreatic endodermcells were generated after exposure with FGF10, RA, CYC, and ILV (d18)and islet-like clusters in presence of HGF, IGF, and GLP-1. Humanpancreas RNA was used as a positive control. No template (water) wasincluded as negative control. (B) Down-regulation ofpluripotency-associated genes upon differentiation. RT-PCR analysis wasperformed to analyze the expression of pluripotency genes (c-MYC, GDF3,hTERT and NANOG) after stepwise differentiation. The same RNA samples asFIG. 8A were used. (C) Formation of islet-like clusters in HCF#1-derivedcells upon differentiation.

iPS-derived pancreatic endoderm were differentiated into islet-likecells with HGF, IGF, DAPT, and GLP-1. Prominent islet-like clusterformation was observed in HCF#1-derived cells. (D) Islet-like clustersexpressed high levels of human C-peptide. (E) Detection of pancreatichormones insulin, C-peptide and glucagon in iPS-derived islet-likecells. Immunofluorescence analysis identified iPS-derived islet-likecells which expressed insulin (green stain was used), C-peptide (redstain was used), and glucagon (red stain was used).

FIG. 9. Sustained PDX1 Expression and Glucose-Responsive C-PeptideSecretion by iPS-derived Islet-like Cells. (A) iPS-derived islet-likecells demonstrated beta cell characteristics. (i) Double-staining ofiPS-derived islet-like cells revealed co-localization of insulin (greenstain was used) and C-peptide (red stain was used), indicating de novoinsulin synthesis. (ii) Some cells were double-positive for insulin(green stain was used) and somatostatin (red stain was used). (iii)Sustained PDX1 expression (red stain was used) in the iPS-derivedinsulin-producing cells after differentiation. Cells were counterstainedwith DAPI. (B) Flow cytometric analysis of iPS-derived islet-like cellsfor insulin expression. iPS-derived islet-like clusters were dissociatedwith TrypLE, and analyzed for insulin expression by an anti-humaninsulin antibody. Insulin staining was observed in HCF#1-derivedislet-like clusters. (C) Glucose-responsive C-peptide secretion by theiPS-derived islet-like clusters. The islet-like clusters weresequentially exposed to low (2.5 mM), intermediate (10 mM), and highconcentrations (27.7 mM) of glucose. Supernatants of HCF#1-derivedislet-like cells were collected and analyzed for C-peptide secretion byELISA. Error bars indicate standard deviation. (D) Glucose-responsiveC-peptide secretion by HCF#1-derived islet-like clusters generated withpancreatogenic cocktails including GLP-1 and ILV (left), GLP-1 withoutILV (middle), or ILV without GLP-1 (right). The islet-like clusters weresequentially exposed to low (2.5 mM), intermediate (10 mM), and highconcentrations (27.7 mM) of glucose. Cumulative C-peptide secretion uponglucose stimulation with 10 mM and 27.7 mM were shown. Error barsindicate standard deviation.

FIG. 10. Formation of Islet-like Clusters in BJ#1 and BJ#SA-derivedCells. iPS-derived pancreatic endoderm cells were differentiated intoislet-like cells with HGF, IGF, DAPT and GLP-1. (A) Islet-like clustersformed in differentiated BJ#1 and BJ#SA cells. (B) Flow cytometricanalysis of iPS-derived islet-like cells for insulin expression.iPS-derived islet-like clusters were dissociated with TrypLE, andanalyzed for insulin expression by an anti-human insulin antibody.Insulin staining was observed in BJ#1 and BJ#SA-derived islet-likeclusters.

FIG. 11. Reprogramming of human hematopoietic progenitor and peripheralblood mononuclear cells. (A) HPCs and PBMCs were cultured in aserum-free medium and transduced with lentiviral vectors expressing fourstemness factors at an MOI of 5. Representative phase-contrast images ofHPCs before transduction (left panel) and 7 day post-infection (leftpanel) are shown. Representative HPC-(left panel) and PBMC-(right panel)derived colonies with characteristic morphologies of reprogrammed cellsare shown. (B) HPC and PBMC-derived iPSC clones were furthercharacterized through immunocytochemistry analysis using a panel ofantibodies against pluripotency-associated markers. All clones stainedpositive for the markers including SSEA-4, TRA-1-60, OCT4, and NANOG.

FIG. 12. Efficient expansion of HSC/PBMC-derived iPSC clones underfeeder- and serum-free conditions. (A) Long-term time-lapse images of aniPSC #HPC-A1 colony were obtained using Nikon BioStation IMQ. Time isshown in hours in the upper right corner, and cell count is shown in thebottom right corner of each panel. (B) Frequent mitotic events wereobserved during time-lapse imaging. Dividing cells and daughter cellsare indicated by downward pointing arrows and upward pointing arrows,respectively. Time is shown in minutes in the upper right corner of eachpanel. (C) High magnification images of a dividing cell at differentstages of mitosis (prophase, prometaphase, metaphase, anaphase, andtelophase) are indicated in arrows. Time is shown in minutes in theupper right corner of each panel.

FIG. 13. Transmission electron microscopic images of blood-derived iPScells. (A) Representative high-resolution electron micrographs ofprimary human fibroblasts (HCF fibroblast), HCF-derived (HCF1 iPS), andHPC-derived (HPC-A1) iPSCs are shown. Mitochondria (MT) and nucleus (N)structures are denoted in the micrographs. (B) Frequent mitotic eventswere observed in the blood-derived iPSCs. Mother and daughter centriolesare represented by the arrowhead and arrow symbols, respectively. Scalebars are represented in μm.

FIG. 14. Global gene expression profiles of blood-derived iPSCs. (A)Dendrogram describing the unsupervised hierarchal clustering of primarykeratinocytes (SW3 HK and SW3 HK), and keratinocyte (HK)-, fibroblast(FB)-, HPC- and PBMC-derived iPSCs. (B) Genome-wide gene expressionpatterns of HPC- and PBMC-derived iPSC clones were compared with thoseof HPCs (GSM178554), PBMCs (GSM452255), verified epidermalkeratinocyte-derived iPSCs (SW4#N1, upper panels), or embryonic stemcells (H9 cells, GSM190779). (C) Heatmap demonstrating the relativeexpression levels ((high-black; low-white) of pluripotency-associatedgenes in primary keratinocytes (HK) and iPS cells from HK, FB or bloodcell sources. The changes in gene expression levels in blood-derivediPSCs, relative to those in HK cells, were calculated using themicroarray data from three primary HK cells and three blood-derivediPSCs, and shown as fold-increase in iPSCs. Statistically significantchanges are indicated by asterisks (p<0.05). (D) Heatmap showing the top100 differentially expressed genes between non-reprogrammed HK andblood-derived iPSC clones (high-black; low-white). Highly expressed innon-reprogrammed cells and blood-derived iPSCs are shown in upper andlower panels, respectively. Genes with notable differences in geneexpression patterns between HK/FB-derived and blood-derived iPSCs areindicated by the gene symbols on the right.

FIG. 15. Differentiation of blood-derived iPSCs in vitro and in vivo.(A) Blood-derived iPSC clones were spontaneously differentiated throughembryoid body formation, and analyzed via immunocytochemistry forlineage markers for three embryonic germ layers (endoderm FOXA2,mesoderm CD31 and ectoderm beta-III-tubulin). (B) Transplantation ofiPSCs into renal capsule of SCID-beige mice resulted in teratomaformation. Tissue histology of teratomas demonstrated the cells of threegerm layers including glandular-, muscular-, and neural rosette-liketissues. (C) Schematic diagram describing the stepwise guideddifferentiation protocol for iPSC differentiation into islet-like cells.DE, definitive endoderm; PG, primitive gut; PE, pancreatic endoderm;ISL, islet-like cells; ActA, Actinin A; Wnt, Wnt3a; FGF10, fibroblastgrowth factor 10; CYC, cyclopamine; RA, all trans retinoic acid; ILV,indolactam V; GLP-1, glucagon-like peptide-1; HGF1, hepatocyte growthfactor-1 and IGF, insulin-like growth factor-1. (D) Through the guideddifferentiation protocol, HSC- or PBMC-derived iPSC clones were inducedto definitive endoderm (day 5), pancreatic endoderm (day 10) andinsulin-producing islet-like cells (day 24). Immunostaining demonstratedthe expression of stage-specific markers in iPSC progeny at day 5 (FOXA2and SOX17), day 10 (NKX6.1 and PDX1) and day 24 (INS). Scale barsindicate 50 μm.

FIG. 16. Expression of pluripotency-associated markers in HK-derived iPSclones. (A) Early-passage HK cells (left panel) were infected withlentivirus (LV) vector encoding OCT4, SOX2, KLF4, and c-MYC. Seven dayspost-infection (center panel), early iPS-like colonies were detected(right panel in higher magnification). (B) HK-derived iPS clones wereeither derived from patients who were non-diabetic (ND) or type 2diabetic (T2D). iPS clones, cultured under feeder-free conditions,exhibited human ES-like morphologies, while expressing high levels ofalkaline phosphatase (AP). (C) Patient HK-derived iPS clones werefurther characterized through immunocytochemistry analysis using a panelof pluripotency markers. All clones were negative for SSEA-1 expression,while staining positive for pluripotency markers SSEA-4, TRA-1-60,TRA-1-81, OCT4, SOX2, KLF4, and NANOG. Scale bars represent 100 μm.

FIG. 17. Pluripotency of HK-derived iPS cells verified throughspontaneous differentiation in vitro and in vivo. (A) HK-derived iPSclones were analyzed via immunocytochemistry for lineage markers forthree germ layers (endoderm, mesoderm and ectoderm). Scale bars indicate50 μm. (B) Transplant of HK-derived iPS cells into the kidney capsule ofSCID-beige mice resulted in teratoma formation. Pictures of harvestedkidneys (with or without iPS transplant) are shown. (C) H&E stainingdemonstrated multiple lineages within the complex architecture of thetumor, including muscle, adipose, immature neuroepithelium, andglandular tissues.

FIG. 18. Variations in gene expression profile upon inducedpluripotency. (A) Dendrogram describing the unsupervised hierarchalclustering of patient-derived HK cells and HK-derived iPS cells. (B)Global gene expression patterns of HK-derived iPS clones were comparedwith their parental HK cells (upper panels), or with that of humanembryonic stem cells (H9, lower panels, GSM190779), upon RNA microarrayanalysis. (C) Heatmap showing the up-regulation and down-regulation(high-black; low-white) of pluripotency-associated genes in HK- andHK-derived iPS clones. The four factors used to induce pluripotency areindicated. The changes in gene expression levels in iPS cells, relativeto those in parental HK cells, were calculated using microarray datafrom three parental HK cells and three HK-derived iPS cells, and shownas fold-induction in iPS cells. Statistically significant changes areindicated by asterisks (p<0.05). HK cells originally expressed highlevels of endogenous KLF4 and c-MYC, resulting in down-regulation ofthese two key reprogramming factors in derived iPS cells. (D) Heatmapshowing the top 15 genes which were up-regulated (upper panel) ordown-regulated (lower panel) upon reprogramming. Statisticallysignificant changes are indicated by asterisks (p<0.05). (E) Comparisonof the major histocompatibility complex (MHC) class I gene expressionprofiles between HK and iPS cells. Statistically significant changes areindicated by asterisks (p<0.05).

FIG. 19. Morphological variations of patient-derived iPS cells uponreprogramming. (A) High-resolution electron micrographs of HK cellsbefore (SW4 parental HK and SW8 parental HK) and after (SW4 #N1, SW3 #B,SW8 #20I, and SW10 #5P) induced pluripotency. Representative micrographof a verified fibroblast-derived iPS cell is also included. Scale barsrepresent 2μm. (B) Mitotic events of two iPS clones were shown (leftpanel in metaphase; right panel in anaphase). Scale bars represent 2μm.(C) Endoplasmic reticulum and the Golgi structures in HK and HK-derivediPS cells are shown. Scale bars represent 0.5 μm. (D) Maturemitochondria with well-developed cristae in parental HK cells (SW8parental) and immature mitochondria in iPS clones (SW3 #B, SW8 #20I, andSW10 #5P) are indicated by arrows. Keratin intermediate filaments inparental HK cells are indicated by arrowheads. Scale bars represent 0.5μm.

FIG. 20. Mitochondrial and oxidative-stress response gene expression ininduced pluripotency. (A) Relative cytochrome B (CYTB) and NADHmitochondrial DNA (mtDNA) copy numbers before (parental) and after (iPS)reprogramming. mtDNA copy numbers were normalized to total genomic DNAand represented as a percentage of parental cell mtDNA copy number. (B)Immunocytochemistry analysis of iPS clone SW4 #N1 with mitochondrialmarker AIF and (C) iPS clones SW4 #N1 and SW10 #5P with MitoTracker(Molecular Probes) staining. (D) Heatmap demonstrating up anddown-regulation of genes involved in mitochondrial biogenesis uponreprogramming (high-black; low-white). No statistically significantchange was observed in any of the genes listed. (E) Heatmap (high-black;low-white) of expression profiles for genes involved in glycolysis,anaerobic glycolysis, and citric acid cycle were compared betweenparental HK and HK-derived iPS cells. Statistically significant changesare indicated by asterisks (p<0.05). (F) RNA expression profiles ofgenes involved in the mitochondrial/oxidative stress response pathwaybetween parental HK and iPS cells are shown. Statistically significantchanges are indicated by asterisks (p<0.05).

FIG. 21. Comparison of telomerase activity, cellular senescence, andprogrammed cell death in HK cells before and after induced pluripotency.(A) RT-PCR analysis of TERT-specific transcripts in parental HK cellsand iPS clones. GAPDH was used as control. (B) Telomere lengths in HKand HK-derived iPS cells were determined by the terminal restrictionfragment lengths. Southern blot analysis and corresponding telomerefragment lengths derived from densitometric quantification are shown.(C) Schematic representation of key senescence- and apoptosis-regulatingpathways. (D) Changes in expression levels of key genes, involved incellular senescence or apoptosis, were determined using the microarraydata of three parental HK cells and three HK-derived iPS cells, and foldinduction of individual genes in iPS cells, relative to those inparental HK cells, are shown. Statistically significant changes areindicated by asterisks (p<0.05).

FIG. 22. Guided in vitro differentiation of patient iPS cells intoinsulin-producing islet-like cells. iPS cells, differentiated throughstep-wise differentiation, were analyzed by immunocytochemistry forstage-specific markers at day 5 (A), 14 (B), 24 (C) and 29 (D and E).Scale bars indicate 50 μm for A, B, C and E (left panel), and 10 μm forD and E (right panel). (F) RT-PCR analysis of the mRNA of SW4#N1 clone,harvested at differentiation day 0, 16, and 29, confirmed the expressionof insulin (INS), glucagon (GCG), somatostatin (SST), and glucosetransporter 2 (GLUT2) on day 29. a-tubulin was used as control (TUBUA).

DETAILED DESCRIPTION

This document provides methods and materials related to differentiatingiPS cells into glucose-responsive, insulin-secreting progeny. Forexample, this document provides methods and material for using ILV andGLP-1 to produce glucose-responsive, insulin-secreting progeny from iPScells.

Any appropriate method can be used to obtain iPS cells. For example, iPScells can be obtained using polypeptides from a species that is the samespecies from which the cells (e.g., somatic cells) were obtained. Anexample of such iPS cells includes human somatic cells that were inducedto form iPS cells using human polypeptides. In some cases, iPS cells canbe obtained using polypeptides from a species that is different from thespecies from which the cells (e.g., somatic cells) were obtained. Anexample of such iPS cells includes human cells that were induced to formiPS cells using mouse polypeptides. Other examples include human cellsthat were induced to form iPS cells using rat, dog, cow, pig, or monkey(e.g., Rhesus monkey) polypeptides. In some cases, an iPS cell providedherein can be a human cell that was induced to form an iPS cell usingnon-human polypeptides (e.g., polypeptides of mouse, rat, pig, dog, ormonkey origin).

The polypeptides used to induce the formation of iPS cells can includeany combination of Oct3/4 polypeptides, Sox family polypeptides (e.g.,Sox2 polypeptides), Klf family of polypeptides (e.g., Klf4polypeptides), Myc family polypeptides (e.g., c-Myc), Nanogpolypeptides, and Lin28 polypeptides. For example, nucleic acid vectorsdesigned to express Oct3/4, Sox2, Klf4, and c-Myc polypeptides can beused to obtain iPS cells. In some cases, Oct3/4, Sox2, Klf4, and c-Mycpolypeptides can be directly delivered into target cells to obtain iPScells using a polypeptide transfection method (e.g., liposome orelectroporation). In one embodiment, nucleic acid vectors designed toexpress Oct3/4, Sox2, and Klf4 polypeptides, and not a c-Mycpolypeptide, can be used to obtain iPS cells. In some cases, Oct3/4,Sox2, and Klf4 polypeptides can be directly delivered into target cellsto obtain iPS cells using a polypeptide transfection method. An Oct3/4polypeptide can have the amino acid sequence set forth in GenBank®Accession Numbers BC117435 (e.g., GI No. 109659099). An Sox2 polypeptidecan have the amino acid sequence set forth in GenBank° Accession NumbersBC013923 (e.g., GI No. 33869633). A Klf4 polypeptide can have the aminoacid sequence set forth in GenBank® Accession Numbers BCO₂₉₉₂₃ (e.g., GINo. 20987475). A c-Myc polypeptide can have the amino acid sequence setforth in GenBank® Accession Numbers BC000141 (e.g., GI No. 12652778). ANanog polypeptide can have the amino acid sequence set forth in GenBank®Accession Numbers BC099704.1 (e.g., GI No. 71043476). A Lin28polypeptide can have the amino acid sequence set forth in GenBank®Accession Numbers BCO₂₈₅₆₆ (e.g., GI No. 33872076).

Any appropriate cell type can be used to obtain iPS cells. For example,skin, lung, heart, liver, blood, kidney, or muscle cells can be used toobtain iPS cells. Such cells can be obtained from any type of mammalincluding, without limitation, humans, mice, rats, dogs, cats, cows,pigs, or monkeys. In addition, any stage of the mammal can be used,including mammals at the embryo, neonate, newborn, or adult stage. Forexample, fibroblasts obtained from an adult human patient can be used toobtain iPS cells. Such iPS cells can be used to treat that same humanpatient (or to treat a different human) or can be used to createdifferentiated cells that can be used to treat that same human patient(or a different human). For example, somatic cells from a human patientcan be treated as described herein to obtain iPS cells. The obtained iPScells can be differentiated into glucose-responsive, insulin-producingcells as described herein that can be implanted into that same humanpatient.

Any appropriate method can be used to introduce nucleic acid (e.g.,nucleic acid encoding polypeptides designed to induce iPS cell formationfrom somatic cells) into a cell. For example, nucleic acid encodingpolypeptides (e.g., Oct3/4, Sox2, Klf4, and c-Myc polypeptides) designedto induce the formation of iPS cells from other cells (e.g.,non-embryonic stem cells or somatic cells) can be transferred to thecells using recombinant viruses that can infect cells, or liposomes orother non-viral methods such as electroporation, microinjection,transposons, phage integrases, or calcium phosphate precipitation, thatare capable of delivering nucleic acids to cells. The exogenous nucleicacid that is delivered typically is part of a vector in which aregulatory element such as a promoter is operably linked to the nucleicacid of interest. The promoter can be constitutive or inducible.Non-limiting examples of constitutive promoters include cytomegalovirus(CMV) promoter and the Rous sarcoma virus promoter. As used herein,“inducible” refers to both up-regulation and down regulation. Aninducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed. The inducer can be achemical agent such as a protein, metabolite, growth regulator, phenoliccompound, or a physiological stress imposed directly by, for exampleheat, or indirectly through the action of a pathogen or disease agentsuch as a virus.

Additional regulatory elements that may be useful in vectors, include,but are not limited to, polyadenylation sequences, translation controlsequences (e.g., an internal ribosome entry segment, IRES), enhancers,or introns. Such elements may not be necessary, although they canincrease expression by affecting transcription, stability of the mRNA,translational efficiency, or the like. Such elements can be included ina nucleic acid construct as desired to obtain optimal expression of thenucleic acids in the cells. Sufficient expression, however, cansometimes be obtained without such additional elements.

Vectors also can include other elements. For example, a vector caninclude a nucleic acid that encodes a signal peptide such that theencoded polypeptide is directed to a particular cellular location (e.g.,the cell surface) or a nucleic acid that encodes a selectable marker.Non-limiting examples of selectable markers include puromycin, adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH),dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase,thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase(XGPRT). Such markers are useful for selecting stable transformants inculture.

Any appropriate viral vectors can be used to introduce stemness-relatedfactors such as Oct3/4, Klf4, Sox2 and c-Myc. Examples of viral vectorsinclude, without limitation, vectors based on DNA or RNA viruses such asadenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses,vaccinia virus, measles viruses, herpes viruses, baculoviruses, andpapilloma virus vectors. See, Kay et al., Proc. Natl. Acad. Sci. USA,94:12744-12746 (1997) for a review of viral and non-viral vectors. Viralvectors can be modified so the native tropism and pathogenicity of thevirus has been altered or removed. The genome of a virus also can bemodified to increase its infectivity and to accommodate packaging of thenucleic acid encoding the polypeptide of interest. In some cases, iPScells can be obtained using viral vectors that do not integrate into thegenome of the cells. Such viral vectors include, without limitation,adenoviral vectors, AAV vectors, baculovirus vectors, and herpesvirusvectors. For example, cells obtained from a human can be providednucleic acid encoding human Oct3/4, Sox2, Klf4, and c-Myc polypeptidesusing viral vectors that do not integrate the exogenous nucleic acidinto the cells. Once the polypeptides are expressed and iPS cells areobtained, the iPS cells can be maintained in culture such that the iPScells are devoid of the exogenous nucleic acid.

Any appropriate non-viral vectors can be used to introducestemness-related factors such as Oct3/4, Klf4, Sox2, and c-Myc. Examplesof non-viral vectors include, without limitation, vectors based onplasmid DNA or RNA, retroelement, transposon, and episomal vectors.Non-viral vectors can be delivered to cells via liposomes, which areartificial membrane vesicles. The composition of the liposome is usuallya combination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Transduction efficiency of liposomes can be increased by usingdioleoylphosphatidylethanolamine during transduction. See, Felgner etal., J. Biol. Chem., 269:2550-2561 (1994). High efficiency liposomes arecommercially available. See, for example, SuperFect® from Qiagen(Valencia, Calif.).

In some cases, iPS cells can be obtained using culture conditions thatdo not involve the use of serum, feeder cells, or serum and feedercells. For example, cells obtained from a human can be provided nucleicacid encoding human Oct3/4, Sox2, Klf4, and c-Myc polypeptides andcultured using media lacking serum (e.g., human or non-human serum) andlacking feeder cells (e.g., human or non-human feeder cells).

Once obtained, iPS cells can be exposed to ILV and GLP-1. For example,human iPS cells can be cultured in the presence of retinoic acid (e.g.,all-trans retinoic acid; RA), an FGF10 polypeptide, KAAD-cyclopamine(CYC), and ILV for a period of time (e.g., about 5 to 15 days, about 6to 15 days, about 5 to 13 days, about 6 to 13 days, about 7 to 12 days,or about 8 to 11 days). After at least about 8 days, the resulting cellscan be cultured in the presence of an hepatocyte growth factor (HGF)polypeptide, an insulin like growth factor (IGF) polypeptide,N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT), and GLP-1 for a period of time (e.g., about 10 to 30 days, about12 to 30 days, about 14 to 30 days, about 10 to 25 days, about 14 to 25days, or about 15 to 24 days) sufficient to result in a population ofglucose-responsive, insulin-secreting cells. In some cases, iPS cells(e.g., human iPS cells) can be cultured in the presence of RA, FGF10,CYC, ILV, HGF, IGF, DAPT, and GLP-1 for a period of time (e.g., about 10to 30 days, about 12 to 30 days, about 14 to 30 days, about 10 to 25days, about 14 to 25 days, or about 15 to 24 days) sufficient to resultin a population of glucose-responsive, insulin-secreting cells.

An FGF10 polypeptide can have the amino acid sequence set forth inGenBank® GI No. 255090638. An HGF polypeptide can have the amino acidsequence set forth in GenBank® GI No. 188595715. A IGF polypeptide canhave the amino acid sequence set forth in GenBank® GI No. 163659904. AGLP-1 polypeptide can have the amino acid sequence set forth in GenBank®Accession Numbers NM_002054.3 (e.g., GI No. 291190799).

Any appropriate amount of these agents (or combination of agents) can beused to obtain glucose-responsive, insulin-secreting cells from iPScells. For example, between about 1 μM and about 3 μM (e.g., about 2 μM)of RA, between about 25 ng/mL and about 75 ng/mL (e.g., about 50 ng/mL)of FGF10 polypeptide, between about 0.2 μM and about 0.3 μM (e.g., about0.25 μM) of CYC, between about 200 nM and about 400 nM (e.g., about 300nM) of ILV, between about 25 ng/mL and about 75 ng/mL (e.g., about 50ng/mL) of HGF polypeptide, between about 25 ng/mL and about 75 ng/mL(e.g., about 50 ng/mL) of IGF polypeptide, between about 5 μM and about15 μM (e.g., about 10 μM) of DAPT, between about 25 nM and about 75 nM(e.g., about 55 nM) of GLP-1 polypeptide can be used together or invarious combinations with culture medium to obtain glucose-responsive,insulin-secreting cells from iPS cells.

Any appropriate method can be used to determine whether or not cellsformed from iPS cells are glucose-responsive, insulin-secreting cells.For example, a C-peptide release assay can be performed to confirm theformation of glucose-responsive, insulin-secreting cells.

Once obtained, the glucose-responsive, insulin-secreting cells can beadministered to a patient to treat, for example, diabetes (e.g., type 1diabetes). For example, iPS-derived pancreatic endoderm cells orglucose-responsive islet-like cells can be transplanted into a humanunder a renal capsule, liver, fat pad, or subcutaneously.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 ILV/GLP-1-Mediated Differentiation of Human iPS Cellsinto Glucose-Responsive Insulin-Secreting Progeny Plasmid Constructionand Lentiviral Vector Production

Stemness factor-expressing lentiviral pSIN-CSGWd1NotI-derived transfervectors were generated as described elsewhere (Nelson et al.,Circulation, 120:408-416 (2009)). In brief, the packaging plasmid pEX-QVwas engineered with H87Q mutation in the HIV-1 capsid region forincreased transduction efficiency of purified infectious supernatants(Nelson et al., Clin. Transl. Sci., 2:118-126 (2009)). HIV vectors wereproduced by transient transfection of 293T cells and titrated byimmunostaining (Nelson et al., Clin. Transl. Sci., 2:118-126 (2009)).Vectors expressed pluripotency factors from a spleen focus-forming virus(SFFV) promoter (Nelson et al., Clin. Transl. Sci., 2:118-126 (2009)).

Generation and Culture of Human iPS Cells on SNL Feeder Cells

Human neonatal foreskin fibroblasts (BJ1) (ATCC#CRL-2522) and primaryhuman cardiac fibroblasts (HCF) (ScienCell #6300) were seeded one daybefore infection in wells of 6 well plates with DMEM containing 10% FBS,Penicillin (100 U/mL) and Streptomycin (100 μg/mL) (Pen/Strep) (completeDMEM). Fibroblasts were infected with lentiviral vectors expressingOCT4, SOX2, KLF4, and c-MYC at a multiplicity of infection about 5 each.After 12 hours of viral infection, cells were fed with fresh completeDMEM. Vector-transduced cells were replated 4 days after infection at5×10⁴ cells per 100 mm dish on mitomycin-C treated SNL feeder cells incomplete DMEM. Next day, the medium was replaced with the serum-freeHEScGRO medium (Millipore #SCM020) supplemented with basic fibroblastgrowth factor (bFGF, 20 ng/mL; Peprotech). Cells were fed with freshHEScGRO medium every two days. Putative iPS colonies, which began toappear 3-4 weeks after vector transduction, were picked based on sizeand human embryonic stem cell-like colony morphology, and expandedthrough dissociation with the cell dissociation buffer (Invitrogen#13151014). BJ1-derived iPS clones, BJ#SA and BJ#SD, were generated onSNL feeder cells. Established iPS clones were maintained in feeder-freecondition.

Feeder Free iPS Generation and Culture

For feeder cell-free iPS generation and maintenance on Matrigel (BDBiosciences #354277)-coated plates, various commercially available stemcell media or their combinations were compared. Optimal results wereobtained when iPS cells were maintained in a feeder cell-free medium,which contained HEScGRO with 25% of mTeSR1 medium (Stemcell Technologies#05850) and 20 ng/mL of bFGF (iPS medium). In order to generatefeeder-cell free iPS clones from BJ and HCF fibroblasts, cells weretransduced with pluripotency factor-expressing lentiviral vectors, 4days after infection. The cells were re-plated at a density of 5×10⁵cells on a Matrigel-coated 100 mm dish. Medium was replaced with freshiPS medium every two days. Putative iPS colonies were observed 1-2 weeksafter vector transduction. iPS clones were picked based on morphologyand size. iPS clones were expanded with cell dissociation buffer andpassaged at a 1:2-1:8 split ratio every 3-7 days depending on celldensity. BJ#1, HCF#1, and HCF#6 iPS clones were generated and maintainedunder feeder cell-free conditions.

RT-PCR

RT-PCR analyses were performed using the primers indicated in Table 1.

TABLE 1RT-PCR primer sequences for human genes analyzed for characterization of human iPScells and differentiation into insulin-producing cells. SEQ SEQ ID IDAccession GI Gene Forward Sequence NO: Reverse Sequence NO: Number No.:OCT4 AGCGAACCAGTATCGAGAAC  1 TTACAGAACCACACTCGGAC  2 BC117435.1109659099 SOX2 AGCTACAGCATGATGCAGGA  3 GGTCATGGAGTTGTACTGCA  4BC013923.2  33869633 NANOG TGAACCTCAGCTACAAACAG  5 TGGTGGTAGGAAGAGTAAAG 6 AB093576.1  31338865 MYC ACTCTGAGGAGGAACAAGAA  7 TGGAGACGTGGCACCTCTT 8 BC000141  12652778 KLF4 TCTCAAGGCACACCTGCGAA  9 TAGTGCCTGGTCAGTTCATC10 BC029923.1  20987475 hTERT TGTGCACCAACATCTACAAG 11GCGTTCTTGGCTTTCAGGAT 12 AB085628.1  22759945 GDF3 AAATGTTTGTGTTGCGGTCA13 TCTGGCACAGGTGTCTTCAG 14 AF263538.1   9652071 FOXA2CTACGCCAACATGAACTCCA 15 AAGGGGAAGAGGTCCATGAT 16 AB028021.1   4958949PDX1 CCCATGGATGAAGTCTACC 17 GTCCTCCTCCTTTTTCCAC 18 U30329.1    929922NEUROG3 GTAGAAAGGATGACGCCTCAACC 19 TCAGTGCCAACTCGCTCTTAGG 20 BC069098.1 46575675 ISL-1 ATTTCCCTATGTGTTGGTTGCG 21 CGTTCTTGCTGAAGCCGATG 22U07559.1    533418 NEUROD1 GAACGCAGAGGAGGACTCAC 23 GTGGAAGACATGGGAGCTGT24 BT019731.1  54696327 NKX6.1 ACACGAGACCCACTTTTTCCG 25TGCTGGACTTGTGCTTCTTCAAC 26 NM_006168.2 111120317 GLUT2GCTACCGACAGCCTATTCTA 27 CAAGTCCCACTGACATGAAG 28 NM_000340.1   4557850MaFA CTTCAGCAAGGAGGAGGTCATC 29 CTCGTATTTCTCCTTGTACAGGTCC 30 NM_201589.2 71274110 INS AGCCTTTGTGAACCAACACC 31 GCTGGTAGAGGGAGCAGATG 32NM_000207.2 109148525 GCG AGGCAGACCCACTCAGTGA 33 AACAATGGCGACCTCTTCTG 34BT006813.1  30582464 SST GTACTTCTTGGCAGAGCTGCTG 35CAGAAGAAATTCTTGCAGCCAG 36 BC032625.1  21619155 GAPDHAGCCACATCGCTCAGACACC 37 GTACTCAGCGGCCAGCATCG 38 BT006893.1  30582624

Immunostaining and Alkaline Phosphatase Staining

For immunostaining, iPS cells were fixed for 20 minutes at roomtemperature (RT) in 4% paraformaldehyde (PFA) in PBS, washed in PBS, andblocked for 30 minutes with 5% FBS in PBST (PBS with 0.1% Tween-20(Sigma). Cells were stained with primary antibodies overnight at 4° C.,rinsed by PBS, and incubated with secondary antibodies 1 hour at RT(Martinez-Fernandez et al., Circ. Res., 105:648-656 (2009)). Cells atdifferent stages of differentiation were fixed and stained with primaryand secondary antibodies. Primary and secondary antibodies used forcharacterization of iPS and derived cells were: SSEA-1, SSEA-4,TRA-1-60, TRA-1-81 (Millipore #SCR001), OCT4 (Cell Signaling Technology#2750), SOX2 (Cell Signaling Technology #2748), KLF4 (Abcam #ab26648),NANOG (Abcam #ab21624), mouse anti-SOX17 (R&D Systems #MAB1924), rabbitanti-HNF3 beta/FOXA2 (Millipore #07-633), rabbit anti-PDX1 (Santa CruzBiotechnology #sc-25403), rabbit anti-NGN3 (Millipore #AB5684), rabbitanti-NEUROD1 (Abcam #16508), mouse anti-insulin (Sigma #12018), rabbitanti-C-peptide (Cell Signaling Technology #4593), rabbit anti-Insulin(Cell Signaling Technology #4590), mouse anti-proinsulin C-peptide(Millipore #CBL94), mouse anti-glucagon (Abcam #ab10988), MafA (SantaCruz Biotechnology #sc-66958), and rabbit anti-somatostatin (Dako#A0566). Texas Red-conjugated donkey-anti-rabbit IgG (JacksonLaboratories #711-075-152), Texas Red conjugated donkey-anti-mouse IgG(Jackson Laboratories #715-075-151), FITC conjugated donkey-anti-rabbitIgG (Jackson Laboratories #711-095-152), and FITC conjugateddonkey-anti-mouse IgG (Jackson Laboratories #715-095-151) were used assecondary antibodies. DAPI was used for counterstaining. Stained cellswere analyzed using confocal laser-scanning microscopy (Zeiss, LSM 510confocal scanning laser system). Alkaline phosphatase staining wasperformed with an Alkaline Phosphatase Detection Kit (Millipore) asdescribed elsewhere (Martinez-Fernandez et al., Circ. Res., 105:648-656(2009)).

Spontaneous Differentiation

For spontaneous differentiation, iPS clones were dissociated usingcollagenase IV and plated on low adhesion plates in basal HEScGRO medium(SCM 021) without bFGF. Embryoid bodies (EBs) were cultured assuspension for 7-14 days and were adherent in knockout DMEM with 20% FBSfor an additional 7-14 days. For immunofluorescence analysis, cells werefixed and stained (Martinez-Fernandez et al., Circ. Res., 105:648-656(2009)). Primary antibodies were: FOXA2 for endoderm, beta III tubulin(Abeam #41489) for ectoderm and CD31 (Santa Cruz Biotechnology #SC1506)for mesoderm, while Texas Red-conjugated donkey-anti-rabbit IgG (JacksonLaboratories #711-075-152) and FITC-conjugated donkey-anti-chicken IgG(Jackson Laboratories #703-095-155) were used as secondary antibodies.

Teratoma Formation and Analysis

A teratoma formation assay was performed using an approved protocol. iPScells were injected subcutaneously into the flank skin of 2-3 months oldathymic nude mice at 500,000 cells/50 μL medium. Tumor growth wasobserved 4-6 weeks after injection. Tumors were processed by rapidfreezing, cut as cryosections, and stained with hematoxylin and eosindyes (Nelson et al., Clin. Transl. Sci., 2:118-126 (2009)).

In Vitro Differentiation of Human iPS Cells to Insulin-SecretingIslet-like Clusters

At the first step of differentiation, human iPS clones were treated with25 ng/mL Wnt3a (R&D systems) and 100 ng/mL activin A (Peprotech) inadvanced RPMI (A-RPMI, Invitrogen) with Pen/Strep for 1 day, followed bytreatment with 100 ng/mL activin A in A-RPMI supplemented with 0.2% FBS(Invitrogen) for two days. At step two, cells were cultured in A-RPMImedium containing 50 ng/mL FGF10 (R&D systems), 0.25 μM KAAD-cyclopamine(CYC), and 2% FBS for 2 days. Cells were then treated with 50 ng/mLFGF10, 0.25 μM CYC, and 2 μM all-trans Retinoic Acid (RA) (Sigma) inDMEM (Invitrogen) supplemented with Pen/Strep, 1×B27 supplement(Invitrogen) for 4 days at step three. Cells were then cultured in thepresence of 50 ng/mL FGF10, 300 nM ILV (Axxora), and 55 nM GLP-1 (Sigma)in DMEM with 1×B27 for 4 days at step four. In step five,differentiation medium included 10 μM DAPT (Sigma) and 55 nM GLP-1 inDMEM with 1×B27 and culture lasted 6 days. Finally, in step six, cellswere cultured in the presence of 50 ng/mL hepatocyte growth factor (HGF)(R&D systems), 50 ng/mL Insulin-like growth factor 1 (IGF-1) (R&Dsystems) and 55 nM GLP-1 in CMRL-1066 medium (Invitrogen) with 1×B27 for6 days. All experiments were repeated more than three times.

C-peptide Content and Glucose Stimulated Secretion Assays

A C-peptide release assay was performed by incubating derived islet-likeclusters in Krebs-Ringer solution with bicarbonate and HEPES (KRBH; 129mM NaCl, 4.8 mM KC1, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5 mMNaHCO₃, 10 mM HEPES, and 0.1% (wt/vol) BSA). Initial incubation wasperformed in KRBH buffer containing 2.5 mM D-glucose for 1 hour at 37°C., followed by incubation in glucose stimulation conditions containing10 mM D-glucose and 27.7 mM D-glucose for 1 hour at 37° C. C-peptide orproinsulin levels were determined using an ultrasensitiveC-peptide/proinsulin ELISA kit (Alpco Diagnostics).

Flow Cytometry

Single-cell suspensions of differentiating human iPS cells were obtainedby dissociating cells with TrypLE (Invitrogen #12605) at 37° C.Intracellular antibody staining was performed using BD Cytofix/Cytopermand BD Perm/Wash buffer. The following antibodies were used:mouse-anti-SOX17 (R&D Systems #MAB1924), guinea pig-anti-insulin (DakoCytomation #A0564), goat-anti-mouse Alexa Fluor 488 (Invitrogen#A11029), and donkey-anti-guinea pig-Cy5 (Jackson ImmunoResearchLaboratories #706-176-148). Flow cytometry data were acquired on aBecton Dickinson FACS Calibur and analyzed using Flowjo software.

Results

Reprogramming of Human Fibroblasts with Stemness Factors

HCF and BJ fibroblasts were infected with lentiviral vectors encodingOCT4, SOX2, KLF4, and c-MYC, and transduced cells re-seeded on mitomycinC-inactivated SNL feeder cells or replated on matrigel-coated plates toensure feeder cell-free culture. On SNL feeder cells, reprogrammedcolonies, characterized by distinct morphology of sharp-edged, flat,tightly-packed structures were visible 2 weeks after viral vectortransduction (FIG. 1A). Under feeder cell-free conditions, similarcolonies were observed as early as day 6 after viral vector infection(FIG. 1B) with clusters of 30-50 cells expressing alkaline phosphatase(FIG. 2). The number of expandable colonies formed on feeders or onnon-feeders plates were 5 to 20 clones per 10⁵ transduced cells.Identified colonies were picked at 3 to 6 weeks to allow sufficientgrowth after viral transduction.

Expression of Pluripotency Markers in Derived iPS Clones

Over 3-9 months or 30-90 passages, putative iPS clones cultured underfeeder cell-free and serum-free conditions exhibited a distinctivemorphology similar to that of human ES cells over long-term culture(FIG. 1C). Tested clones expressed high levels of alkaline phosphatase(FIG. 1D). Immunocytochemistry revealed expression of SSEA-4, TRA-1-60,TRA-1-81, OCT4, SOX2, KLF4, and NANOG in multiple clones (FIGS. 3A and3B). These clones were negative for SSEA-1 expression. RT-PCR of totalcellular RNA further demonstrated induction of endogenouspluripotency-associated genes, including OCT4, SOX2, GDF3, telomerase(TERT), KLF4, c-MYC, and NANOG (FIG. 3C). No notable difference wasobserved between clones isolated from BJ and HCF fibroblasts, or withclones isolated with SNL feeder cells. Morphology and expression of stemcell genes indicated establishment of human iPS clones from fibroblasts,and maintenance in an undifferentiated state under feeder-freeconditions.

Pluripotency Validated through Three Germ Layer Differentiation

Human iPS clones were assayed, through embryoid body (EB) formation, forthe ability to spontaneously differentiate in vitro into cells of thethree embryonic germ layers. All iPS clones assayed formed EBs (FIG.4A). After variable times in suspension, EBs were transferred toadherent conditions and further cultured. Immunostaining forlineage-specific markers confirmed that human iPS cells differentiatedinto ectoderm (beta-III tubulin, FIG. 4A), endoderm (FOXA2, FIG. 4A) andmesoderm (CD31, FIG. 4A) lineages. Moreover, in vivo human iPS cellsformed teratomas after injection into nude mice. These subcutaneoustumors enlarged up to 1 cm in diameter within 3 months post-injection(FIG. 4B). Histology revealed diverse cell types, including glandularepithelium (ectoderm, FIG. 4C), adipose (endoderm, FIG. 4C) and muscular(mesoderm, FIG. 4C) tissues. Thus, human iPS cells generated from BJ andHCF fibroblasts exhibit hallmark properties of pluripotent stem cells.

Differentiation of Human iPS cells into Pancreatic Endoderm

Normal differentiation of a pluripotent precursor into lineage-specifiedpancreatic endodermal tissue encompasses multiple steps. Here, verifiediPS cells were treated first with activin A and Wnt3a for generation ofdefinitive endoderm cells, and then with FGF10 and CYC for derivation ofgut tube endoderm (FIG. 5A). Derived cells were further treated withFGF10, RA, and CYC in the absence or presence of ILV for generation ofpancreatic endoderm, followed by culture in HGF, IGF, and DAPT in theabsence or presence of GLP-1 for generation of pancreatichormone-expressing cells (FIG. 5A). In this way, human iPS clones wereinduced to form definitive endoderm by treatment with activin A andWnt3a initially for 1 day followed by culture in activin A and 2% FBSfor 2 additional days. Immunostaining of treated cells revealedefficient SOX17 and FOXA2 induction, markers of definitive endoderm(FIG. 5B). Similar results were observed with clones generated fromhuman cardiac fibroblasts or foreskin (FIG. 6). Flow cytometrydemonstrated that 92%, 72%, and 84% cells were positive for SOX17 inthree distinct clones, respectively (FIGS. 5C and 6). Next, theefficiency of definitive endoderm transformation into pancreaticendoderm was evaluated. Initial attempts to generate pancreatic endodermby stimulating definitive endoderm cells with FGF10 and CYC for twodays, followed by FGF10, RA, and CYC stimulation resulted in cells withlow levels of PDX1 expression (data not shown). ILV, which is describedelsewhere (Chen et al., Nat. Chem. Biol., 5:258-265 (2009) and Borowiaket al., Cell Stem Cell, 4:348-358 (2009)), was included in the protocol.In the protocol, treatment of iPS-derived definitive endoderm cells withFGF10, RA, and CYC in the presence of ILV resulted in cells expressingPDX1, NEUROD1, and NGN3, markers of pancreatic endoderm (FIG. 5D).Similar results were observed for iPS cells derived from human cardiacfibroblasts or foreskin (FIG. 7). These results demonstrate thesuccessful induction of pancreatic endoderm from iPS-derived definitiveendoderm.

Induction of Stage-Specific Pancreatic Genes through GuidedDifferentiation

To determine the expression of endocrine-specific transcription factorsand pancreas-specific genes throughout differentiation, the geneexpression pattern was analyzed at each stage of differentiation. RT-PCRdetected high levels of FOXA2 expression after 3 days ofdifferentiation, confirming induction of definitive endoderm cells (FIG.8A). The expression of the endocrine progenitor-specific gene, NGN3, wasobserved from day 3 of differentiation, and the expression persistedthroughout the differentiation process (FIG. 8A). Expression of theislet specific gene, ISL-1, was also found from day 3, with expressionlevels increasing at later time points (FIG. 8A). Moreover, PDX1 andNEUROD1 transcripts, which were found only after treatment with FGF10,RA, CYC, and ILV, further confirmed the generation of iPS-derivedpancreatic endoderm cells upon differentiation (FIG. 8A). To evaluatewhether human iPS-derived pancreatic endoderm cells are capable ofgenerating functional pancreatic islet-like cells, an additional step ofdifferentiation was used. The iPS-derived pancreatic endoderm wereinitially treated with HGF, IGF, Exendin-4, and DAPT; however, resultingcells failed produce detectable C-peptide secretion (data not shown).GLP-1, which is described elsewhere (Buteau et al., Diabetes, 52:124-132(2003)) was included. Following inclusion of GLP-1, RT-PCR revealedpositive gene expression of pancreatic hormones, including insulin,glucagon (GCG), and somatostatin (SST), and islet cell-specific markergenes PDX1, NKX6.1, ISL1, and NEUROD1 and glucose transporter 2 (GLUT2)(FIG. 8A). Conversely, to determine whether pluripotency genes weresilenced during differentiation, RT-PCR analysis was performed forc-MYC, GDF3, hTERT, NANOG, SOX2, and KLF4. It was found that c-MYC,GDF3, hTERT, and NANOG gene expression levels gradually decreased duringdifferentiation, while these gene transcripts were absent in the humanpancreas (FIG. 8B). SOX2 and KLF4 gene expression remained throughoutiPS differentiation, in line with expression of these two genes in thehuman pancreas (FIG. 8B). The targeted down-regulation of pluripotencygenes along with sequential expression of pancreas-specific genescollectively indicated that human iPS cells are capable of undergoingguided differentiation in vitro into islet-like cells, with the observedcombined expression of GLUT-2, NKX6.1, and NEUROD1 further suggestingderivation of tissue with properties of functional beta cells.

Differentiation of iPS Cells into Insulin-Secreting Islet-like Progeny

During treatment with RA, FGF10, CYC, and ILV, iPS-derived pancreaticendoderm cells started to form spheroid-like cell clusters, whichreached maximum size and number following further maturation with HGF,IGF, DAPT, and GLP-1 (FIG. 8C). The three dimensional morphologyresembled pancreatic islet-like clusters (Ramiya et al., Nat. Med.,6:278-282 (2000)), and selected clones yielded clusters (FIG. 8C)strongly positive for C-peptide expression (FIG. 8D). Importantly, eveniPS-derived islet-like cells that did not organize into typical clustersalso expressed insulin, C-peptide, and glucagon (FIG. 8E). The presenceof insulin/C-peptide co-expressing cells (FIG. 9A (i)) confirmed thepotential for de novo insulin synthesis and excluded the possibleartifact of insulin uptake from the media. Also, insulin-glucagon doublepositive cells were not observed, indicating that the expression patternof iPS-derived hormone-expressing islet-like cells is consistent withnormal pancreatic beta-cell development. Although few insulin andsomatostatin double-positive cells were found (FIG. 9A(ii)),characteristic of immature islet cells, the results provided hereinindicate successful differentiation of iPS cells into hormone-expressingislet-like cells. Indeed, similar to pancreatic beta cells, whichco-express insulin and PDX1, the majority of the insulin-expressingcells exhibited nuclear-localized PDX1 signals (FIG. 9A (iii)). When theinsulin-positive population was quantified by flow cytometry, 1.3%,0.7%, and 0.8% of distinct clones-derived islet-like cells wereinsulin-positive (FIGS. 9B and 10).

Functional Response of iPS-derived Islet-like Clusters

C-peptide secretion from iPS-derived islet-like clusters in response toglucose challenge, the critical physiological function of pancreaticbeta cells, was analyzed. To determine whether islet-like cells arecapable of C-peptide secretion in response to glucose induction, cellswere exposed to increasing concentrations of glucose and secretedC-peptide was measured by ELISA. At extracellular glucose levels of 2.5mM, that mimics a fasting condition, there was only marginal detectionof the C-peptide signal (FIG. 9C). Raising glucose levels to 10 mMinduced marked secretion of C-peptide by iPS-derived islet-like cells(FIG. 9C). Further raising glucose levels to the supraphysiological 27.7mM range, triggered an additional bolus of secreted C-peptide, reachingcumulatively the range of 72.0-236.1 pM (HCF#1, three independentexperiments, FIG. 9C) or 12.1-50.9 pM (BJ#1). iPS-derived islet-likecells differentiated without ILV or GLP-1 failed to secrete C-peptide inresponse to glucose challenge (FIG. 9D). Although clonal variation inresponsiveness was observed, iPS-derived islet-like clusters weretypically capable to secrete C-peptide in response to glucosestimulation.

Example 2 Induced Pluripotent Stem Cells from GMP-Grade HematopoieticProgenitor Cells and Monocytes Cells

Clinical grade peripheral blood hematopoietic progenitor cells (HPC)products from patients, who were deceased, were used. HPCs wereharvested from patients following mobilization by injection withgranulocyte-CSF for 5 days. Blood (10-20 L) was processed for HPCcollection. PBMCs from healthy donors were obtained as describedelsewhere (Noser et al., J. Virol., 80:7769-7774 (2006)).

Lentiviral Vector Production

Pluripotency-associated factor-expressing lentiviral vectors, pSIN-OCT4,pSIN-SOX2, pSIN-KLF4, and pSIN-cMYC, were described elsewhere (Nelson etal., Clin. Transl. Sci., 2:118-126 (2009)). These vectors were producedby transient transfection of 293T cells. Vector titers were determinedby immunostaining (Nelson et al., Clin. Transl. Sci., 2:118-126 (2009)).

iPSC Derivation

HPCs and PBMCs were cultured overnight in StemSpan H3000 serum-freemedium (StemCell Technologies), which contained only human-derived orrecombinant human proteins, supplemented with StemSpan CC100 cytokinecocktail (StemCell Technologies). Cultures were then transduced withfour stemness factor-expressing lentiviral vectors overnight. One thirdof the culture supernatants were carefully removed and replaced dailywith H3000 growth medium supplemented with CC100 cytokine cocktail. At 3days after vector infection, cells were transferred to Matrigel (BDBioscience)-coated culture plates. Starting 5 days after vectorinfection, cells were maintained in HEScGRO medium (100 mL, Millipore)supplemented with mTeSR-1 maintenance media (25 mL, StemcellTechnologies) (Thatava et al., Gene Ther., 18:283-293 (2011)). Seven toten days after vector infection, the reprogrammed cells began to formcolonies with iPS morphology. At two to three weeks after vectorinfection, cultures were treated with Cell Dissociation Buffer(Invitrogen) for 5 to 10 minutes to help lift clones, and individualiPSC-like clones were carefully picked up by a P200 pipette and placedinto Matrigel-coated wells in a 96-well plate. To prevent spontaneousdifferentiation, the iPSC culture medium was replaced daily, anddifferentiated cells in the cultures were manually removed with apipette tip. As the clones grew, cultures were expanded into largerculture plates for further characterization. Clones were preserved usingXeno-FREEze™ Human Embryonic Stem Cell Freezing Medium (Millipore). Averified iPSC clone, HCF1, from primary human fibroblast (HCF) cells,was described elsewhere (Thatava et al., Gene Ther., 18:283-293 (2011)).Primary human keratinocytes and keratinocyte-derived iPSC clones werealso used as controls.

Immunostaining and Alkaline Phosphatase Staining

For immunostaining of iPSC, cells were fixed for 20 minutes at roomtemperature in 4% paraformaldehyde solution in PBS, washed several timesin PBS, and blocked for 30 minutes in PBS with 5% fetal bovine serum.Cells were then stained with primary antibodies overnight at 4° C.,rinsed by PBS, and incubated with secondary antibodies for 1 hour atroom temperature. For immunostaining of differentiated cells, cells atdifferent stages of differentiation were fixed and stained with primaryand secondary antibodies. Primary antibodies used for characterizationof iPSC and iPSC-derived cells were: SSEA-4 and TRA-1-60 (Millipore#SCR001), OCT4 (Cell Signaling Technology #2750), NANOG (Abcam#ab21624), mouse anti-SOX17 (R&D Systems #MAB1924), rabbit anti-HNF3beta/FOXA2 (Millipore #07-633), rabbit anti-PDX1 (Santa CruzBiotechnology#sc-25403), and mouse anti-insulin (Sigma #12018). TexasRed-conjugated donkey-anti-rabbit IgG (Jackson Laboratories#711-075-152), Texas Red-conjugated donkey-anti-mouse IgG (JacksonLaboratories #715-075-151), FITC-conjugated donkey-anti-rabbit IgG(Jackson Laboratories #711-095-152), and FITC-conjugateddonkey-anti-mouse IgG (Jackson Laboratories #715-095-151) were used assecondary antibodies. DAPI was used for counter staining. Stained cellswere analyzed using confocal laser-scanning microscope (Zeiss, LSM 510confocal scanning laser system).

Spontaneous Differentiation

For spontaneous differentiation, iPSC clones were dissociated usingcollagenase IV for 30 minutes and plated on low adhesion plates in basalHEScGRO medium without bFGF. Embryoid bodies (EBs) were cultured assuspension for 7-10 days and adherent in DMEM with 20% FBS foradditional 7-10 days. For immunofluorescence analysis, cells were fixedwith 4% PFA for 20 minutes at room temperature. Immunostaining wasperformed as described above. Primary antibodies against FOXA2 forendoderm, beta-III tubulin (Abcam #41489) for ectoderm, and CD31 (SantaCruz Biotechnology #SC1506) for mesoderm were used, while TexasRed-conjugated donkey anti-rabbit IgG (Jackson Laboratories#711-075-152), and FITC-conjugated donkey anti-chicken IgG (JacksonLaboratories #703-095-155) served as secondary antibodies.

In Vivo Differentiation of Derived iPS Cells

SCID-beige mice were anesthetized, and the kidney was externalized foriPS transplantation under the kidney capsule. A small incision was madein the kidney capsule, and a blunt needle was used to create a pocketunder the kidney capsule. Following iPSC injection into the pocket, thekidney was placed back into the abdomen, and the incision closed withvicryl suture. Mice were maintained for 4 weeks and sacrificed forharvesting normal and iPS-transplanted kidneys. OTC-embedded frozentissues were cryo-sectioned for H&E staining.

Differentiation of Derived iPS Cells into Insulin-Producing Cells

iPSC were differentiated into insulin-producing cells as reportedelsewhere with several modifications (Thatava et al., Gene Ther.,18:283-293 (2011)). At the first step of differentiation, human iPSCclones were treated with 25 ng/mL Wnt3a (R&D systems) and 100 ng/mLactivin A (Peprotech) in advanced RPMI (Invitrogen) with Pen/Strep for 1day, followed by treatment with 100 ng/mL activin A in advanced RPMIsupplemented with 0.2% fetal calf serum (FCS) (Invitrogen) for two days.At step two, cells were cultured in high glucose DMEM

(Invitrogen), supplemented with 20% (v/v) advanced RPMI mediumcontaining 50 ng/mL FGF10 (R&D systems), 0.25 μM KAAD-cyclopamine (CYC),and 2% FCS for 2 days. Cells were then treated with 50 ng/mL FGF10, 0.25μM CYC, and 2 μM all-trans Retinoic Acid (RA) (Sigma) in high glucoseDMEM (Invitrogen) supplemented with 20% advanced RPMI, Pen/Strep, 1×B27supplement (Invitrogen) for 4 days at step three. Cells were thencultured in the presence of 50 ng/mL FGF10, 300 nM ILV (Axxora), and 55nM GLP-1 (Sigma) in DMEM (high glucose) supplemented with 20% advancedRPMI and 1×B27 for 4 days at step four. In step five, differentiationmedium included 10 μM DAPT (Sigma) and 55 nM GLP-1 in DMEM (highglucose) with 20% advanced RPMI and 1×B27 and culture lasted 6 days.Finally, in step six, cells were cultured in the presence of 50 ng/mLhepatocyte growth factor (HGF) (R&D systems), 50 ng/ml insulin-likegrowth factor 1 (IGF-1) (R&D systems), and 55 nM GLP-1 in CMRL-1066medium (Invitrogen) with 1×B27 for 8 days. All differentiationexperiments were performed in triplicate, and repeated at least twotimes.

Microarray

Total RNA was isolated using TRIzol (Invitrogen) and further purifiedusing RNeasy Plus spin columns (QIAGEN). Turbo DNA-free DNase (Ambion,Austin, Tex.) was used to digest all genomic DNA that could lead tofalse positive gene expression results. The RNA quantity and purity wasmeasured with a Nanodrop spectrophotometer (Thermo Scientific,Wilmington, Del.), and the RNA integrity was determined using theAgilent 2100 Bioanalyzer (Santa Clara, Calif.). Microarray analysis wasperformed using the Affymetrix HG-U133 Plus2 GeneChip Array platform(Affymetrix, Santa Clara, Calif.). Data were preprocessed using standardin-house MicroArray Pre-Processing workflow, and hierarchical clusteringwas performed by Pearson Dissimilarity. To compare the transcriptome ofblood-derived iPSCs, the data set of epidermal keratinocytes (HK, SW3,SW4 and SW8), two keratinocyte-derived iPSC clones (SW3 #b and SW4 #N1),and human fibroblast (FB)-derived iPSC clone HCF1 (Thatava et al., GeneTher., 18:283-293 (2011)) were used. T-test was performed to analyze thesignificance of the changes (p<0.05) in the normalized gene expressionlevels between HK and iPSC clones, or between blood-derived iPSC clonesand HK- and FB-derived iPSC clones. Heatmap Builder software (providedby Dr. Euan Ashley, Stanford School of Medicine) was used to generate aheatmap for the transcriptome data set. The registered GEO transcriptomedatabase (GSM551202, human ES H9 cells; GSM452255, freshly isolatedPBMC; GSM178554, mobilized HPCs) were used to analyze the similaritiesbetween blood-derived iPSC and human ES cells or non-reprogrammed PBMCsand HPCs.

Results

Cellular Reprogramming of HPCs and PBMCs into iPSCs

HPCs and PBMCs were cultured overnight in a serum-free medium with CC100cytokine cocktail (recombinant Flt-3, SCF, IL-3 and IL-6), andtransduced with four stemness factor-expressing lentiviral vectors at anMOI of 5 each. When transduced cells were transferred to Matrigel-coatedculture plates at day 3 post-infection, a subset of cells attached tothe plate. At 1 to 2 weeks after vector transduction, small,reprogrammed colonies, characterized by the morphology of sharp-edged,flat and tightly-packed cells, were observed (FIG. 11A). No iPSC-likecolony formation was observed in untransduced cells (FIG. 11A).Individual iPSC-like colonies were picked based on their size andmorphology at 2 to 3 weeks after viral transduction and expanded underfeeder-free conditions. The number of iPS-like colonies, expandedwithout substantial spontaneous differentiation, was between 2 to 10clones per 10⁵ transduced cells (FIG. 11B). HPC- and PBMC-derived iPSclones were capable of being cultured for 5 months after the initialvector infection (up to passage 50) without showing signs of replicativecrisis. Immunocytochemistry revealed the expression of SSEA-4, TRA-1-60,OCT4, and NANOG in the blood-derived iPSC clones (FIG. 11B). Long-termtime-lapse imaging demonstrated efficient iPSC expansion underfeeder-free and serum-free conditions, with a 23.7 hour average celldoubling time (FIG. 12A). Frequent mitotic events were observed inderived iPSC colonies (FIG. 12B), and the duration of mitotic events(from prophase to telophase) was approximately 60 minutes (FIGS. 12B and12C).

Ultrastructural Studies of Blood-Derived iPS Cells

High-resolution electron microscope analysis was performed to determinethe morphological differences between blood-derived iPSCs and verifiedfibroblast-derived iPSCs (HCF1) (Thatava et al., Gene Ther., 18:283-293(2011)). Blood-derived iPSCs exhibited scant cytoplasm andglobular-shaped immature mitochondria with unorganized cristae, whichresembled those of fibroblast-derived iPS cells (FIG. 13A). In contrast,non-reprogrammed fibroblasts exhibited the cytoplasm densely packed withmembrane-bound organelles (FIG. 13A, upper left panel) including maturemitochondria with well-developed cristae (FIG. 13A, upper right panel).In accordance with the cinemicrography analysis, frequent mitotic eventswere observed in blood-derived iPSCs cells (FIG. 13B). One pair ofcentrioles—mother (arrowhead) and daughter (arrow) centrioles—were seenin a dividing cell at anaphase (FIG. 13B, lower right panel).

Genome-Wide Transcriptome Analysis of Blood-Derived iPS Clones

Using a microarray representing the genome-wide transcriptome, theglobal gene-expression patterns in HPC- and PBMC-derived iPSC cloneswere determined, which were then compared with those of fibroblast (FB)-and epidermal keratinocytes (HK)-derived iPSCs. Transcriptome data fromnon-reprogrammed HK cells were also used as somatic cell controls. Thedendrogram of unsupervised one-way hierarchical clustering analysisdemonstrated that blood-derived iPSCs clustered closely with other iPSCsfrom different cell sources and were distinct from non-reprogrammed HKcells (FIG. 14A). In accordance with this observation, the globalgene-expression patterns of blood-derived iPSCs were more similar tothose in human ES H9 cells and HK-derived iPSCs, rather thannon-reprogrammed HSCs or PBMCs (FIG. 14B). Similar to HK- and FB-derivediPSC clones, expression of pluripotency-associated genes, such as OCT4,SOX2, NANOG, LIN28, and TERT, were markedly up-regulated in HPC- andPBMC-derived iPSC clones (FIG. 14C). When the top 100 differentiallyexpressed genes between blood-derived iPSC clones and non-reprogrammedHK cells were analyzed and used to generate heatmaps including FB- andHK-derived iPS cells, the gene expression patterns of blood-derivediPSCs were nearly identical to those of iPSCs derived from FB and HKcells. Among the 200 differentially expressed genes (100 highest and 100lowest), notable differences in gene expression profiles were only foundin XIST (with three probes, FIG. 14D, upper panel), USP9Y, EIF1AY,DDX3Y, and RPS4Y1 (FIG. 14D, lower panel) in two HK-derived iPSC clones(SW3 #b and SW3 #NI). XIST is on the X chromosome and XIST RNA plays amajor role in silencing one of the pair of X chromosomes in female cells(Nagano and Fraser, Cell, 145:178-181 (2011)), while USP9Y, EIF1AY,DDX3Y, and RPS4Y1 are Y-linked genes. Since HK and HK-derived iPSCclones were from female patients, while HCF1, HPC-A1, PBMC-S1 andPBMC-S2 were from male patients, the observed variations in X- andY-linked genes between blood- and non-blood-derived iPSC clones werelikely due to the difference in gender of these iPSC clones.

Pluripotency of Blood-Derived iPS Clones Verified Through In VitroDifferentiation

HPC- and PBMC-derived iPSC clones were assayed for the ability tospontaneously differentiate in vitro into cells of three embryonic germlayers through embryoid body (EB) formation. All the iPSC clones assayedformed EBs. After 7 to 10 days in suspension, EBs were transferred to aMatrigel-coated plate, and spontaneously differentiated cells wereexpanded under adherent conditions. Immunostaining for lineage-specificmarkers revealed that blood-derived iPSCs differentiated into cells ofthree germ layers including beta-III tubulin-positive ectoderm,FOXA2-positive endoderm, and CD31-positive mesoderm cells (FIG. 15A).

In Vivo Multilineage Differentiation of Blood-Derived iPSCs

To assess the multilineage differentiation capacity of iPSCs in vivo,blood-derived iPSCs were transplanted under the kidney capsule ofSCID-beige mice. Following transplantation of 1 million cells, iPSCsformed cystic tumors within 4 weeks (FIG. 13B). Upon gross inspection,iPSC-derived tumors demonstrated a complex cellular architecture withprominent vascularization and nonvascularized solid tissues.Histological analysis revealed iPSC differentiation into endodermlineages composed of glandular-like tissue, mesoderm lineages indicatedby muscle-like tissue and ectoderm lineages denoted by neuralrosette-like structures (FIG. 15B), which verified the multi-lineagedifferentiation capability of blood-derived iPSCs.

Generation of Insulin Producing Cells from iPSCs through GuidedDifferentiation

The pancreatic differentiation potentials of blood-derived iPSCs wasexamined. A guided iPSC differentiation protocol with indolactam V (ILV)and GLP-1 was used as set forth above. Blood-derived iPSC clones werefirst stimulated with actin A and Wnt3a to form definitive endodermcells. Immunostaining revealed the efficient induction of definitiveendoderm markers SOX17 and FOXA2 in iPSC-derived cells at day 5 ofdifferentiation (FIG. 13C). Derived definitive endoderm cells werefurther differentiated in DMEM/advanced RPMI medium containing FGF10,CYC, and 2% FBS (v/v) for 2 days, and maintained in high glucoseDMEM/advanced RPMI medium supplemented with FGF10, CYC, RA, and 1 x B27for an additional 4 days. Cells were then cultured in the presence ofFGF10, ILV, GLP-1, and 1×B27 in DMEM/advanced RPMI medium for 4 days.After this step, derived cells expressed pancreatic endoderm markers,PDX1 and NKX6.1 (FIG. 15D). Further differentiation of iPSC-derivedpancreatic endoderm cells was performed in DMEM/advanced RPMI mediumsupplemented with DAPT, GLP-1, and 1×B27 for 6 days, followed by thefinal maturation step in the CMRL-1066 medium containing HGF, IGF-1,GLP-1, and 1×B27 for an additional 8 days. Insulin-positive iPSC progenywere sporadically detected (FIG. 15D). High levels of intracellularC-peptide (230-320 pM), a byproduct of proinsulin processing duringinsulin secretion, were also detected in the final differentiation stageiPSC progeny by C-Peptide ELISA. These results demonstrate successfuldifferentiation of blood-derived iPSCs into insulin-expressing cells invitro.

The results provided herein demonstrate the feasibility of iPSCderivation from GMP-grade mobilized HPCs and unmobilized PBMCs. The useof HPCs and PBMCs enabled time-effective iPSC derivation, as the cellsdid not require long-term expansion before reprogramming. Moreover,apart from minor differences in global gene expression profiles (FIG.14), blood-derived iPSCs were basically indistinguishable from iPSCsfrom other cell sources. Considering that many institutes/hospitalsalready have FDA-approved GMP facility for autologous HPC processing,HPCs and PBMCs can be used as described herein as ideal somatic cellsources for clinical-grade iPSC derivation.

The results provided herein also demonstrate the feasibility ofgenerating insulin-producing cells from blood-derived iPSCs. In contrastto skin biopsies, which involve an invasive procedure, the use of bloodcells allows minimally invasive tissue procurement for iPSC derivation.Since diabetic patients often experience poor wound healing, theminimally invasive iPSC derivation from blood cell sources would beparticularly advantageous for the generation of clinical-grade iPSCsfrom diabetic patients.

Example 3 Reprogrammed Keratinocytes from Elderly Type 2 DiabetesPatients Suppress Senescence Genes to Acquire Induced Pluripotency HumanKeratinocytes

Skin specimens from surgical pathology from nondiabetic and type 2diabetic (T2D) individuals were enzymatically processed. Using steriletechniques, skin samples were incubated overnight at 4° C. in dispase(25 U/mL) to cleave epidermis from dermis. The epidermal layer was thenplaced into a recombinant trypsin/EDTA solution (Invitrogen, Carlsbad,Calif.) and incubated for 30 min at 37° C. Trypsin/EDTA was neutralizedwith a trypsin inhibitor (Invitrogen, Carlsbad, Calif.), and epidermalpieces were pipetted to release epidermal cells. The suspension was thenpassed through a 70 μm cell strainer and pelleted. Cell viability wasdetermined by the trypan blue exclusion method. Cells were seeded in aplate coated with an animal component-free (ACF) coating matrix(Invitrogen). Selective trypsinization removed fibroblasts at about 6minutes, while human keratinocytes (HK) were dissociated at about 20minutes. HK cell populations were then grown in EpiLife Medium and S7growth supplement (Invitrogen, Carlsbad, Calif.) in 5% CO₂ and 95% airat 37° C. HK cells were maintained semi-confluent in low calcium media.

Reprogramming

Lentiviral vectors, pSIN-OCT4, pSIN-SOX2, pSIN-KLF4, and pSIN-cMYC, weremanufactured as described elsewhere to express pluripotency factors froman internal spleen focus-forming virus (SFFV) promoter (Nelson et al.,Clin. Transl. Sci., 2:118-126 (2009)). HIV vectors were produced bytransient transfection of 293T cells. To minimize calcium-mediateddifferentiation of HK cells during vector infection, lentiviral vectorswere concentrated by ultracentrifugation and re-suspended in PBS (Sakumaet al., Hum. Gene Ther., 21:1665-1673 (2010)). Lentiviral titers weredetermined by immunostaining (Nelson et al., Clin. Transl. Sci.,2:118-126 (2009)). Human HK cells were grown in vitro in ACF EpiLifeMedium in a matrix-coated plate. Cultures were transduced overnight withhuman OCT4, SOX2, KLF4, and cMYC expressing lentiviral vectors (Nelsonet al., Clin. Transl. Sci., 2:118-126 (2009)). Culture supernatants werereplaced daily with ACF media. At 4 days after vector infection, mediawas changed to HEScGRO medium (100 mL, Millipore, Billerica, Mass.)supplemented with mTeSR-1 maintenance media (25 mL, StemcellTechnologies, Vancouver, BC, Canada) (Thatava et al., Gene Ther.,18:283-293 (2011)). One to two weeks after vector infection,reprogrammed cells began to form colonies displaying stem cellmorphology (Thatava et al., Gene Ther., 18:283-293 (2011)). At three tofour weeks after vector infection, cultures were treated with CellDissociation Buffer (Invitrogen, Carlsbad, Calif.) for 5 to 10 minutesto help lift clones picked by a P200 pipette, and placed in BD Matrigel(BD Biosciences, San Jose, Calif.) coated 96-well plates. To preventspontaneous differentiation, the iPS culture medium was replaced dailyand differentiated cells in cultures manually removed. As clones grew,cultures were expanded into larger culture plates for furthercharacterization. iPS clones were preserved using Xeno-FREEze™ HumanEmbryonic Stem Cell Freezing Medium (Millipore, Billerica, Mass.). Forspontaneous differentiation, iPS clones were dissociated usingcollagenase IV (Stemcell Technologies) for 30 minutes and plated on lowadhesion plates in basal HEScGRO medium without bFGF. Embryoid bodies(EBs) were cultured as suspensions for 7-14 days, and grown adherent inDMEM with 20% FBS for additional 7-14 days.

Differentiation of iPS Cells into Insulin Producing Cells

iPS clones were treated with 25 ng/mL Wnt3a (R&D systems) and 100 ng/mLactivin A (Peprotech) in advanced RPMI (Invitrogen) with Pen/Strep for 1day, followed by treatment with 100 ng/mL activin A in advanced RPMIsupplemented with 0.2% fetal calf serum (FCS) (Invitrogen) for two days.Next, cells were cultured in high glucose DMEM (Invitrogen),supplemented with 20% (v/v) advanced RPMI medium containing 50 ng/mLFGF10 (R&D systems), 0.25 μM KAAD-cyclopamine (CYC), and 2% FCS for 2days. Cells were then treated with 50 ng/mL FGF10, 0.25 μM CYC, and 2 μMall-trans Retinoic Acid (RA) (Sigma) in high glucose DMEM (Invitrogen)supplemented with 20% advanced RPMI, Pen/Strep, 1×B27 supplement(Invitrogen) for 4 days. Cells were then cultured in 50 ng/mL FGF10, 300nM ILV (Axxora), and 55 nM GLP-1 (Sigma) in DMEM (high glucose)supplemented with 20% advanced RPMI and 1×B27 for 4 days.Differentiation medium including 10 μM DAPT (Sigma) and 55 nM GLP-1 inDMEM (high glucose) with 20% advanced-RPMI and 1×B27 was used to culturecells for the next 6 days. Finally, cells were cultured in 50 ng/mLhepatocyte growth factor (HGF) (R&D systems), 50 ng/mL insulin-likegrowth factor 1 (IGF-1) (R&D systems), and 55 nM GLP-1 in CMRL-1066medium (Invitrogen) with 1×B27 for 8 days.

Immunostaining

For immunostaining, iPS cells were fixed for 20 minutes at roomtemperature in 4% paraformaldehyde (PFA), washed in PBS, and blocked for30 minutes in PBST (PBS with 0.1% Tween-20 (Sigma) and 5% FBS). Cellswere stained with primary antibodies overnight at 4° C., rinsed by PBS,and incubated with secondary antibodies for 1 hour at room temperature.Separately, cells at different stages of differentiation were fixed andstained with primary and secondary antibodies. Primary and secondaryantibodies used for characterization were: SSEA-1, SSEA-4, TRA-1-60,TRA-1-81 (Millipore #SCR001), OCT4 (Cell Signaling Technology #2750),SOX2 (Cell Signaling Technology #2748), KLF4 (Abcam #ab26648), NANOG(Abcam #ab21624), anti-SOX17 (R&D Systems #MAB1924), anti-HNF3beta/FOXA2 (Millipore #07-633), anti-PDX1 (Santa CruzBiotechnology#sc-25403), and anti-insulin (Sigma #12018). TexasRed-conjugated anti-rabbit IgG (Jackson Laboratories #711-075-152),Texas Red-conjugated anti-mouse IgG (Jackson Laboratories #715-075-151),FITC-conjugated anti-rabbit IgG (Jackson Laboratories #711-095-152), andFITC-conjugated anti-mouse IgG (Jackson Laboratories #715-095-151) wereused as secondary antibodies. DAPI was used to counter-stain nuclei.Stained cells were analyzed using confocal laser-scanning microscopy(Zeiss, LSM 510 confocal scanning laser system). Alkaline phosphatasestaining was performed with an Alkaline Phosphatase Detection Kit(Millipore). Antibodies FOXA2 for endoderm, beta III tubulin (Abcam#41489) for ectoderm and CD31 (Santa Cruz Biotechnology #SC1506) formesoderm were used to immunostain embryoid body-derived cells.

In Vivo Differentiation of iPS Cells

SCID-beige mice were anesthetized, and the kidney exposed for iPStransplantation under the kidney capsule. To this end, a small incisionwas made in the kidney capsule, and a blunt needle was used to create apocket under the kidney capsule. Following iPS cell injection, thekidney was placed back into the abdomen, and the incision closed. Micewere maintained for 4 weeks and sacrificed for harvesting normal andiPS-transplanted kidneys. OTC-embedded frozen tissues werecryo-sectioned for H&E staining.

Gene Expression

For amplification of mitochondrial DNA, mitochondria-specific primerpairs (CYTB, CCTAGCCATGCACTACTCACCAGACGCCT (SEQ ID NO:39),CTGTCTACTGAGT-AGCCTCCTCAGATTC (SEQ ID NO:40); and NADH,TCACCAAAGAGCCCCTAA-AACCCGCCACATCTA (SEQ ID NO:41),TAAGGGTGGAGAGGTTAAAGGAGC (SEQ ID NO:42)) were used. For RT-PCR analysis,total RNA was isolated using TRIzol (Invitrogen), and reversetranscription was performed with oligo (dT) primer using RNA to cDNAEcoDry (Clontech). Platinum Taq DNA polymerase (Invitrogen) and primerpairs for TERT (TGTGCACCAACATCTACAAG (SEQ ID NO:43),GCGTTCTTGGCTTTCAGGAT (SEQ ID NO:44)), INS (AGCCTTTGTGAACCAACACC (SEQ IDNO:45), GCTGGTAG-AGGGAGCAGATG (SEQ ID NO:46)), SST(GTACTTCTTGGCAGAGCTGCTG (SEQ ID NO:47), CAGAAGAAATTCTTGCAGCCAG (SEQ IDNO:48)), GCG (AGGCAGACC-CACTCAGTGA (SEQ ID NO:49), AACAATGGCGACCTCTTCTG(SEQ ID NO:50)), GLUT2 (GCTACCGACAGCCTATTCTA (SEQ ID NO:51),CAAGTCCCACTGACATGAAG (SEQ ID NO:52)), and a-tubulin(AAGAAGTCCAAGCTGGAGTTC (SEQ ID NO:53), GTTG-GTCTGGAATTCTGTCAG (SEQ IDNO:54)) were used for the reaction. Separately, total RNA was isolatedusing TRIzol (Invitrogen) and further purified using RNeasy Plus spincolumns (QIAGEN). Turbo DNA-free DNase (Ambion, Austin, Tex.) was usedto digest all genomic DNA that could lead to false positive geneexpression results. RNA quantity and purity were measured with aNanodrop spectrophotometer (Thermo Scientific, Wilmington, Del.), andRNA integrity was determined using the Agilent 2100 Bioanalyzer (SantaClara, Calif.).

Microarray analysis was performed using the Affymetrix HG-U133 Plus2GeneChip Array platform (Affymetrix, Santa Clara, Calif.). Data werepreprocessed using MicroArray Pre-Processing workflow, and hierarchicalclustering was performed by Pearson Dissimilarity. For comparison oftranscriptome data between pre- and post-reprogramming, the data set ofparental HK cells from three patients (SW3, SW4 and SW8) were comparedwith those of three iPS clones from the same patients (SW3 #B, SW4 #N1,and SW8 #20I). Student's t-test was performed to assess significance(p<0.05) in normalized gene expression levels between HK and HK-derivediPS clones. The Heatmap Builder software (provided by Dr. Euan Ashley,Stanford University) was used to generate the heatmap for thetranscriptome data set. Enrichment analysis was also performed to matchgene IDs in functional ontologies. The registered GEO transcriptomeinformation (GSM551202, human ES H9 cell transcriptome) was used asreference.

Telomere Assay

Total genomic DNA was isolated from patient-derived HK and iPS cellsusing QIAGEN

DNeasy Blood & Tissue Kit. Telomere length was determined usingTeloTAGGG telomere length assay (Roche). Genomic DNA digestion, Southernblotting, and chemiluminescence detection was performed as perestablished protocols. Densitometric analysis was performed on AdobePhotoshop, and terminal restriction fragment lengths were determined byΣ(OD_(i))/Σ(OD_(i)/L), where OD_(i) and L were the optical density andlength of fragment, respectively.

Results Reprogramming of Human Keratinocytes

Lentiviral vectors encoding human OCT4, SOX2, KLF4, and c-MYC, at anapproximate multiplicity of infection of 5 each, transduced earlypassage human keratinocytes (HK cells) derived from 56 to 78 year-oldindividuals with or without T2D. Under serum-free and feeder-freeconditions, within 1 to 2 weeks after viral vector infection, smallreprogrammed colonies, characterized by a sharp-edged, flat,tightly-packed morphology, were apparent (FIG. 16A). Individual colonieswere picked based on size and morphology at 3 to 5 weeks after viraltransduction, and expanded. Structurally derived clones resembled humanES or fibroblast-derived iPS cells and expressed high levels of thestemness marker alkaline phosphatase (FIG. 16B). Immunocytochemistryfurther validated robust expression of diverse pluripotency markers,including SSEA-4, TRA-1-60, TRA-1-81, OCT4, SOX2, KLF4, and NANOG inHK-derived iPS clones regardless of patient age and status of diabetes(FIG. 16C). The obtained yield was 2 to 10 expandable clones per 10⁵transduced cells with maintained pluripotent markers and absence ofreplicative crisis even at 7 months post-initial vector infection (up topassage 60).

Differentiation Propensity of Derived iPS Cells

HK-derived iPS clones from diabetic and non-diabetic patientsspontaneously differentiated in vitro into cells of all three germlayers within embryoid body (EB) formations (FIG. 17). In line withacquired pluripotency, HK-derived iPS cells differentiated into ectoderm(beta-III tubulin), endoderm (FOXA2), and mesoderm (CD31) as detected byimmunostaining for lineage-specific markers (FIG. 17A). Clonal, ratherthan inter-patient, variations in differentiation propensities wasobserved within the tested cohort (FIG. 17A). Moreover, in vivoHK-derived iPS cells transplanted under the kidney capsule of SCID-beigemice at a dose of 1 million cells gave rise to 1-2 cm outgrowth within 4weeks (FIG. 17B). Tissue histology revealed iPS differentiation intomesoderm lineages indicated by muscle and adipocytes (FIG. 17C),ectoderm lineages denoted by neuroepithelium-like tissues (FIG. 17C),and endoderm lineages composed of glandular tissue (FIG. 17C). Thesedata document multilineage propensity of HK-derived iPS cells from bothdiabetic and non-diabetic patients across tested age groups.

Genome-Wide Transcriptome Switch Underlies Transition to InducedPluripotency

Unbiased scan of the genome-wide transcriptome revealed distinct globalgene-expression patterns in parental HK versus HK-derived iPS clones(FIG. 18). The dendrogram of unsupervised one-way hierarchicalclustering analysis demonstrated that HK-derived iPS cells fromdifferent patients clustered together, and branched out from parentalorigin (FIG. 18A). Consistent with acquisition of a pluripotenttranscriptome, gene expression patterns of HK-derived iPS cells wereoverall similar to those of human ES H9 cells, and different fromparental counterparts (FIG. 18B). Induction of key pluripotency genes,such as OCT4, SOX2, NANOG, LIN28, telomerase (TERT), DPPA4, and PODXL,were also evident in iPS clones (FIG. 18C). Further analysis revealedupon reprogramming significantly up-regulated proto-oncogenes (N-MYC andKIT), pluripotency-maintenance factor FGF-2, and the receptor for FGF-2(FGFR1), whereas cytoskeletal and keratin-encoding genes weredown-regulated across HK-derived iPS clones (FIG. 18D). Similar to EScells, which are known to express minimal levels of MHC class I genes,HK-derived iPS cells exhibited marked down-regulation of these genes(FIG. 18E). Bioinformatic analysis of transcriptome data identifiedpathways involved in epithelial-to-mesenchymal transition andcytoskeletal remodeling as most significantly affected networks inresponse to reprogramming of HK cells, in line with genuine redirectionof cell fate. No notable difference was observed in the transcriptome ofiPS clones from non-diabetic and diabetic patients.

Ultrastructural Remodeling Induced by Reprogramming

Electron microscopy demonstrated marked difference in the size ofderived iPS compared to parental HK (FIG. 19). Parental HK cells were 25to 40 μm in diameter, while derived iPS cells were 10 to 15 μm,characterized by scant cytoplasm and regularly condensed chromatin (FIG.19A) with frequent mitotic events (FIG. 19B). The cytosol of HK cellswas densely packed with membrane-bound organelles (FIG. 19C, left panel)and keratin intermediate filaments. In sharp contrast, widelydistributed, relatively poorly developed endoplasmic reticulum and Golgistacks were found in iPS clones (FIG. 19C, right panel). In HK cells,mitochondria appeared mainly tubular-shaped and showed well-developedcristae. In contrast, mostly globular immature mitochondrial remnants,characterized by unorganized cristae, were found in HK-derived iPS cells(FIG. 19D) as in verified fibroblast-derived iPS clones (FIG. 19A). Nonotable difference was observed in morphologies of mitochondria betweeniPS clones from non-diabetic and diabetic patients.

Reprogramming Down-Regulates Mitochondria/Oxidative Stress SignalingPathway

The copy number of mitochondrial DNA before and after reprogrammingrevealed a 30 to 60% reduction in the abundance of mitochondrial DNA iniPS compared to HK cells (FIG. 20A). Immunostaining with mitochondrialprobes detected mitochondria-specific signals in individual iPS cells(FIG. 20B and 20C) and no significant changes in expression ofnuclear-encoded mitochondrial biogenesis factors (FIG. 20D). Selectedgenes involved in the TCA cycle, such as ACO_(2,) SDHA, and FH, weredown-regulated by nuclear reprogramming (FIG. 20E). Transcriptomeanalysis further revealed that genes encoding themitochondrial/oxidative stress response pathway are highly expressed inHK cells from elderly patients, yet markedly down-regulated in derivediPS cells (FIG. 20F). Reduced transcription following reprogramming wasparticularly evident in major antioxidant enzymes (Finkel et al.,Nature, 408:239-247 (2000)), such as catalase CAT and GPX1 (FIG. 20F),suggesting reversal of cellular markers of senescence.

Reprogramming Induces Telomere Elongation and Down-Regulates GenesInvolved in Senescence

RT-PCR verified increased levels of TERT-specific transcripts inHK-derived iPS cells (FIG. 21A). In fact, the telomere restrictionfragment (TRF) assay further demonstrated that HK-derived iPS cell linesdisplay longer telomeres than parental HK cells (FIG. 21B), indicatingreprogramming induced telomere elongation regardless of diabetes status.Comparison of the transcriptome between three parental HK cells (SW3-HK,SW4-HK, and SW8-HK) and derived iPS clones (SW3 #B, SW4 #N1, and SW8#20I) revealed significant down regulation (p <0.05) ofsenescence/apoptosis-associated genes (FIG. 21C), including p16^(INK4a)and p15^(INK4b) in the p16^(INK4a)/RB pathway, and p21^(CIP1) in thep19^(ARF)/p53 pathway, and proapoptotic genes, including FAS, CASP8,CASP7, BAD, and TP53AIP1 (FIG. 21D). These results indicated thatsuccessful cellular reprogramming of somatic cells from elderly patientsis associated with suppression of key senescence- and apoptosis-relatedpathways in diabetic and non-diabetic patients.

Proficiency of HK-Derived iPS Cells to Yield Insulin ProducingIslet-Like Progeny

HK-derived iPS clones were initially induced to form definitive endodermby treatment with activin A and Wnt3a for 1 day followed by culture inactivin A and 2% FBS for 4 additional days. Immunostaining revealedefficient induction in iPS-derived cells of SOX17 and FOXA2, markers ofdefinitive endoderm (FIG. 22A). Similar results were observed with iPSclones generated from diabetic or non-diabetic patients. Next, theefficiency of definitive endoderm transformation into pancreaticendoderm was evaluated. As shown in FIG. 22B, prominentnucleus-localized signals for pancreatic endoderm, namely PDX1 andNKX6.1, were found in iPS-derived cells at day 14 of differentiation. Nonotable difference was found among iPS clones from non-diabetic anddiabetic patients. These results indicate successful induction ofpancreatic endoderm from HK-iPS-derived definitive endoderm. In thepresence of DAPT and GLP-1, iPS-derived pancreatic endoderm cells werefurther differentiated for 6 days, followed by maturation in HGF, IGF-1,and GLP-1 for additional 8 days. By day 24, insulin-producing cells weresporadically detected in iPS-derived progeny (FIG. 22C), while moreprominent immunostaining for insulin was evident after final maturationat day 29 (FIG. 22D and 22E). Similar to pancreatic beta cells whichco-express insulin and PDX1, the majority of iPS-derivedinsulin-expressing cells exhibited nuclear-localized PDX1 signals (FIG.22D and 22E). High levels of intracellular C-peptide (250-290 pM), abyproduct of proinsulin protein processing, were detected in iPS progenyby ELISA, while RT-PCR revealed positive gene expression of keypancreatic factors, including insulin (INS), glucagon (GCG), andsomatostatin (SST), and glucose transporter 2 (GLUT2) (FIG. 22F). Thus,HK-derived iPS cells differentiated into hormone-producing pancreaticislet-like cells.

These results demonstrate the feasibility and reproducibility of iPScell derivation from elderly patients with T2D. Reprogramming of HKcells was accompanied by morphological changes, induction of endogenouspluripotency genes, telomere elongation, and down-regulation ofsenescence- and apoptosis-related genes. Notably, stepwisedifferentiation with ILV and GLP-1 achieved successfully differentiationof T2D-specific iPS cells into insulin-producing islet-like cells. Thus,reprogramming of keratinocytes from elderly T2D patients yieldsproficient iPS cells through induction of a senescence privilegedstatus. T2D-specific iPS cells can provide a versatile platform fordisease modeling and regenerative applications.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method for obtaining a population ofglucose-responsive, insulin-secreting cells from a human, wherein saidmethod comprises: (a) obtaining somatic cells from a human, (b) exposingsaid somatic cells to one or more polypeptides or nucleic acids encodingsaid one or more polypeptides to form induced pluripotent stem cells,wherein said one or more polypeptides are selected from the groupconsisting of a Oct3/4 polypeptide, a Sox family polypeptide, a Klffamily polypeptide, a Myc family polypeptide, a Nanog polypeptide, and aLin28 polypeptide, and (c) culturing said induced pluripotent stem cellswith medium comprising indolactam V and glucagon like peptide-1 toobtain said population of glucose-responsive, insulin-secreting cells.3. The method of claim 2, wherein said medium lacks serum.
 4. The methodof claim 2, wherein said culturing is performed in the absence of feedercells.
 5. The method of claim 2, wherein said culturing is performed inthe absence of non-human feeder cells.
 6. The method of claim 2, whereinsaid somatic cells are selected from the group consisting of skin, lung,heart, stomach, brain, liver, blood, kidney, and muscle cells.
 7. Themethod of claim 2, wherein said induced pluripotent stem cells compriseexogenous nucleic acid encoding a human Oct4 polypeptide, a human Sox2polypeptide, a human Klf4 polypeptide, and a human c-Myc polypeptide. 8.The method of claim 2, wherein said medium comprises between about 200nM and about 400 nM of indolactam V.
 9. The method of claim 2, whereinsaid medium comprises between about 25 nM and about 75 nM of glucagonlike peptide-1.
 10. The method of claim 2, wherein said culturing isperformed for about 10 to 25 days.